Compositions and methods of use for augmented immune response and cancer therapy

ABSTRACT

The present invention provides antibody compositions, including, e.g., antibodies, engineered antibodies and antibody fragments that bind to a tumor necrosis factor receptor superfamily member (i.e., 18), and compositions comprising one or more additional therapeutic agents. Provided compositions are useful in enhancing CD4+ and CD8+ T cell responses, and in the treatment, amelioration and prevention of diseases that can be counteracted with an augmented immune response, e.g., cancers. Also provided are methods of use of combinations that find use in treatment or prevention of cancerous or infectious conditions and disorders.

RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. Provisional Application No. 62/061,644, filed Oct. 8, 2014, U.S. Provisional Application No. 62/198,673, filed Jul. 29, 2015, and U.S. Provisional Application No. 62/220,764, filed Sep. 18, 2015, each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies, antibody fragments, and antigen binding molecules that bind to tumor necrosis factor receptor superfamily member 18/glucocorticoid induced TNFR-related protein (“GITR”), and more specifically that are agonists, stimulate signaling through the receptor and/or modulate immune response.

BACKGROUND OF THE INVENTION

Glucocorticoid-induced TNFR-related protein (“GITR”) is a member of the Tumor Necrosis Factor Superfamily (TNFRSF) which includes more than 20 type I transmembrane proteins, several splicing variants and several viral proteins, all of which have a cysteine-rich domain as a common structural feature. The extracellular domain (ECD) of GITR consists of 3 cysteine-rich domains (CRDs), followed by a transmembrane domain (TM) and an intracellular domain (ICD).

GITR expression is detected constitutively on murine and human CD4+CD25+ regulatory T cells which can be further increased upon activation. In contrast, effector CD4+CD25− T cells and CD8+CD25− T cells express low to undetectable levels of GITR, which is rapidly upregulated following T cell receptor activation. Expression of GITR has also been detected on activated NK cells, dendritic cells, and macrophages. Signal transduction pathway downstream of GITR has been shown to involve MAPK and the canonical NFκB pathways. Various TRAF family members have been implicated as signaling intermediates downstream of GITR (Nocentini et al. (2005) Eur. J. Immunol., 35:1016-1022).

Cellular activation through GITR is believed to serve several functions depending on the cell type and microenvironment including, but not limited to, costimulation to augment proliferation and effector function, inhibition of suppression by regulatory T cells, and protection from activation-induced cell death (Shevach and Stephens (2006) Nat. Rev. Immunol., 6:613-618). Ko et al. ((2005) J. Exp. Med., 202:885-891) first demonstrated that an agonistic monoclonal antibody against mouse GITR effectively induced tumor-specific immunity and eradicated established tumors in a mouse syngeneic tumor model. Additionally and/or alternatively, an anti-mGITR which has functional Fc effector activity has been shown in some preclinical models to deplete regulatory T cells, as well as enhance T effector cell proliferation and cytokine secretion in select tumor environment. These findings suggest that an agonistic antibody to mGITR can disrupt immune tolerance balance, which in turn will allow T cells to combat tumors and persistent viral infections. However, studies to date have largely focused on use of surrogate antibodies in rodent systems. Due to the divergence of structure among mouse and human GITR, it is unknown whether findings seen with surrogate studies in mouse would translate to modification of human GITR function.

DESCRIPTION OF THE INVENTION

We have identified antibodies that specifically bind to human glucocorticoid-induced tumor necrosis factor receptor superfamily member 18 (“GITR”), wherein the antibodies have in vitro hGITR agonist activity when cross-linked in vitro, and wherein the antibodies confer hGITR activity in vivo and induce an elevated Teff:Treg ratio at tumor sites, resulting in inhibition of tumor progression. Thus, the present invention provides agonist antibodies, antibody fragments, and antigen binding molecules that specifically bind to and promote intracellular signaling and/or modulate immune response through targeting cells expressing human GITR. In one aspect, the invention provides isolated antibodies, antibody fragments, and antigen binding molecules that specifically bind to human GITR, wherein the antibody, antibody fragment, or the antigen binding molecule binds to an epitope comprising the cysteine-rich domain 1 (“CRD1”, SEQ ID NO:4: CGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDC) and the cysteine-rich domain 2 (“CRD2”, SEQ ID NO:5: MCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQC), and wherein the antibody, antibody fragment, or the antigen binding molecule is an agonist of GITR, and wherein the antibody, antibody fragment, or the antigen binding molecule optionally has an intact or increased FcR effector function.

In some embodiments, the antibody, antibody fragment, or the antigen binding molecule binds to an epitope comprising SEQ ID NO:88) of human GITR. In some embodiments, the antibody, antibody fragment, or antigen binding molecule competes with an antibody or antibody fragment that binds to an epitope comprising SEQ ID NO:88 of human GITR. In some embodiments, the antibody, antibody fragment, or antigen binding molecule binds to at least one amino acid residue within SEQ ID NO:88 of human GITR, for example, the antibody, antibody fragment, or antigen binding molecule binds to an epitope that overlaps with SEQ ID NO:88 of human GITR.

In some embodiments, the antibody, antibody fragment, or the antigen binding molecule binds to an epitope comprising CRD1 (residues 34-72, SEQ ID NO:4) and residue 78 of human GITR. In some embodiments, the antibody, antibody fragment, or antigen binding molecule competes with an antibody or antibody fragment that binds to an epitope within CRD1 (residues 34-72, SEQ ID NO:4) and residue 78 of human GITR. In some embodiments, the antibody, antibody fragment, or antigen binding molecule binds to at least one amino acid residue within CRD1 (residues 34-72, SEQ ID NO:4) and residue 78 of human GITR, for example, the antibody, antibody fragment, or antigen binding molecule binds to an epitope that overlaps with CRD1 (residues 34-72, SEQ ID NO:4) and residue 78 of human GITR.

In some embodiments, the antibody, antibody fragment, or antigen binding molecule binds to SEQ ID NO:1 and comprises (a) a heavy chain variable region comprising a human heavy chain wherein: i) the heavy chain CDR1 comprises SEQ ID NO:22, and ii) the heavy chain CDR2 comprises a sequence selected from any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, and iii) the heavy chain CDR3 comprises SEQ ID NO:29 or SEQ ID NO:109; and (b) a light chain variable region, wherein i) the light chain CDR1 comprises SEQ ID NO:30 or SEQ ID NO:31, and ii) the light chain CDR2 comprises SEQ ID NO:33, and iii) the light chain CDR3 comprises SEQ ID NO:34.

In some embodiments, the antibody, antibody fragment, or antigen binding molecule binds to SEQ ID NO:88 and comprises (a) a heavy chain variable region comprising a human heavy chain wherein: i) the heavy chain CDR1 comprises SEQ ID NO:22, and ii) the heavy chain CDR2 comprises a sequence selected from any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, and iii) the heavy chain CDR3 comprises SEQ ID NO:29 or SEQ ID NO:109; and (b) a light chain variable region, wherein i) the light chain CDR1 comprises SEQ ID NO:30 or SEQ ID NO:31, and ii) the light chain CDR2 comprises SEQ ID NO:33, and iii) the light chain CDR3 comprises SEQ ID NO:34.

With respect to further embodiments of the antibodies, antibody fragments, or antigen binding molecules, in some embodiments the heavy chain variable region has at least 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the variable region of SEQ ID NO:16 and the light chain variable region has at least 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the variable region of SEQ ID NO:17. In particular embodiments, the antibody, antibody fragment, or antigen binding molecule comprises a heavy chain comprising SEQ ID NO:16 and a light chain comprising SEQ ID NO:17. In some embodiments, the antibody, antibody fragment, or antigen binding molecule competes with an antibody that comprises a heavy chain comprising SEQ ID NO:16 and a light chain comprising SEQ ID NO:17.

In some embodiments, the heavy chain FR4 is a human germline FR4. In particular embodiments, the heavy chain FR4 is SEQ ID NO:42.

In some embodiments, the light chain FR4 is a human germline FR4. In particular embodiments, the light chain FR4 is SEQ ID NO:50.

In some embodiments, provided is an antibody, antibody fragment or antigen binding molecule wherein: i) the heavy chain CDR1 comprises SEQ ID NO:22 or SEQ ID NO:84; ii) the heavy chain CDR2 comprises SEQ ID NO:28 or SEQ ID NO:80; iii) the heavy chain CDR3 comprises SEQ ID NO:29 or SEQ ID NO:109; iv) the light chain CDR1 comprises SEQ ID NO:30 or SEQ ID NO:85; v) the light chain CDR2 comprises SEQ ID NO:33 or SEQ ID NO:82, and vi) the light chain CDR3 comprises SEQ ID NO:34 or SEQ ID NO:83.

In some embodiments, provided is an antibody, antibody fragment or antigen binding molecule wherein: wherein: i) the heavy chain CDR1 comprises SEQ ID NO:22; ii) the heavy chain CDR2 comprises SEQ ID NO:23; iii) the heavy chain CDR3 comprises SEQ ID NO:29; iv) the light chain CDR1 comprises SEQ ID NO:30; v) the light chain CDR2 comprises SEQ ID NO:33, and vi) the light chain CDR3 comprises SEQ ID NO:34.

In some embodiments, provided is an antibody, antibody fragment or antigen binding molecule wherein: wherein: i) the heavy chain CDR1 comprises SEQ ID NO:22; ii) the heavy chain CDR2 comprises SEQ ID NO:24; iii) the heavy chain CDR3 comprises SEQ ID NO:29; iv) the light chain CDR1 comprises SEQ ID NO:31; v) the light chain CDR2 comprises SEQ ID NO:33, and vi) the light chain CDR3 comprises SEQ ID NO:34.

In some embodiments, provided is an antibody, antibody fragment or antigen binding molecule wherein: wherein: i) the heavy chain CDR1 comprises SEQ ID NO:22; ii) the heavy chain CDR2 comprises SEQ ID NO:25; iii) the heavy chain CDR3 comprises SEQ ID NO:29; iv) the light chain CDR1 comprises SEQ ID NO:30; v) the light chain CDR2 comprises SEQ ID NO:33, and vi) the light chain CDR3 comprises SEQ ID NO:34.

In some embodiments, provided is an antibody, antibody fragment or antigen binding molecule wherein: wherein: i) the heavy chain CDR1 comprises SEQ ID NO:22; ii) the heavy chain CDR2 comprises SEQ ID NO:26; iii) the heavy chain CDR3 comprises SEQ ID NO:29; iv) the light chain CDR1 comprises SEQ ID NO:30; v) the light chain CDR2 comprises SEQ ID NO:33, and vi) the light chain CDR3 comprises SEQ ID NO:34.

In some embodiments, provided is an antibody, antibody fragment or antigen binding molecule wherein: wherein: i) the heavy chain CDR1 comprises SEQ ID NO:22; ii) the heavy chain CDR2 comprises SEQ ID NO:27; iii) the heavy chain CDR3 comprises SEQ ID NO:29; iv) the light chain CDR1 comprises SEQ ID NO:30; v) the light chain CDR2 comprises SEQ ID NO:33, and vi) the light chain CDR3 comprises SEQ ID NO:34.

In some embodiments, provided is an antibody, antibody fragment or antigen binding molecule wherein: wherein: i) the heavy chain CDR1 comprises SEQ ID NO:22; ii) the heavy chain CDR2 comprises SEQ ID NO:25; iii) the heavy chain CDR3 comprises SEQ ID NO:109; iv) the light chain CDR1 comprises SEQ ID NO:30; v) the light chain CDR2 comprises SEQ ID NO:33, and vi) the light chain CDR3 comprises SEQ ID NO:34.

In a further aspect, the invention provides antibodies, antibody fragments, or antigen binding molecules that specifically bind GITR, wherein the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region wherein: i) the CDR1 of the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO: 79, or SEQ ID NO:84; ii) the CDR2 of the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:62, and SEQ ID NO:80; iii) the CDR3 of the heavy chain comprises SEQ ID NO:29 or SEQ ID NO:109; iv) the CDR1 of the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:63, SEQ ID NO:81, SEQ ID NO:85, and SEQ ID NO:86; v) the CDR2 of the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:64, and SEQ ID NO:82; and the CDR3 of the light chain comprises SEQ ID NO:34 or SEQ ID NO:83.

In other embodiments of the antibodies, antibody fragments, or antigen binding molecules, the heavy chain variable region has at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the variable region of a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:99 and SEQ ID NO:105, and the light chain variable region has at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the variable region of a sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:7. In particular embodiments, the isolated antibody, antibody fragment, or antigen binding molecule comprises a heavy chain variable domain comprising a sequence selected from any of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:99 and SEQ ID NO:105; and light chain variable domain comprising SEQ ID NO:7 or SEQ ID NO:9. In some embodiments, the isolated antibody, antibody fragment, or antigen binding molecule comprises a heavy chain variable domain of SEQ ID NO:6 and a light chain variable domain of SEQ ID NO:7. In some embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising SEQ ID NO:8 and a light chain variable domain comprising SEQ ID NO:9. In other embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising SEQ ID NO:10 and a light chain variable domain comprising SEQ ID NO:7. In other embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising SEQ ID NO:12 and a light chain variable domain comprising SEQ ID NO:7. In other embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising SEQ ID NO:14 and a light chain variable domain comprising SEQ ID NO:7.

With respect to further embodiments of the antibodies, antibody fragments, or antigen binding molecules, in some embodiments the heavy chain variable region has at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the variable region of SEQ ID NO:99 and the light chain variable region has at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the variable region of SEQ ID NO:7. In some embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising the SEQ ID NO:99 and a light chain variable domain comprising SEQ ID NO:7.

With respect to further embodiments of the antibodies, antibody fragments, or antigen binding molecules, in some embodiments the heavy chain variable region has at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the variable region of SEQ ID NO:105 and the light chain variable region has at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the variable region of SEQ ID NO:7. In some embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising the SEQ ID NO:105 and a light chain variable domain comprising SEQ ID NO:7.

In some embodiments, the antibody, antibody fragment, or antigen binding molecule that binds to GITR is humanized. In certain embodiments, the antibody or antibody fragment comprises a human constant region.

In some embodiments, the antibody fragment is a Fab′ fragment. In some embodiments, the antibody fragment is a single chain antibody (scFv). In some embodiments, the antibody fragment is a single-domain antibody or nanobody.

In some embodiments, the antibody or antibody fragment is cross-linked to a second antibody or antibody fragment. In some embodiments, the antibody is glycosylated.

In some embodiments, the antibody, antibody fragment, or antigen binding molecule is an IgG. In certain embodiments the antibody, antibody fragment, or antigen binding molecule comprises an IgG isotype antibody Fc region. In particular embodiments the antibody, antibody fragment, or antigen binding molecule comprises an IgG1 or an IgG2 isotype antibody Fc region. In certain embodiments the antibody, antibody fragment, or antigen binding molecule is an IgG1 or an IgG2 antibody. In some embodiments, the antibody, antibody fragment, or antigen binding molecule contains at least one mutation that modulates (i.e., increases or decreases) binding of the antibody or antibody fragment to an Fc receptor. In some embodiments, the antibody, antibody fragment, or antigen binding molecule contains at least one mutation that modulates (i.e., increases or decreases) the antibody, antibody fragment, or antigen binding molecule to activate an Fc receptor. In particular embodiments, the antibody, antibody fragment, or antigen binding molecule contains at least one mutation that increases binding of the antibody or antibody fragment to an Fc receptor. In certain embodiments, the antibody, antibody fragment, or antigen binding molecule contains at least one mutation that increases the antibody, antibody fragment, or antigen binding molecule to activate an Fc receptor.

In some embodiments, the antibody, antibody fragment, or antigen binding molecule cross-reacts with human and non-human primate GITR. In some embodiments, the antibody, antibody fragment, or antigen binding molecule does not cross-react with rodent GITR, e.g., rat GITR or mouse GITR.

In a related aspect, the invention further provides polynucleotides encoding an antibody, antibody fragment or antigen binding molecule of the invention as described herein. In some embodiments, the polynucleotide encoding the light chain variable region has at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a nucleic acid sequence selected from SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:102. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a nucleic acid sequence selected from SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:101, and SEQ ID NO:107. In some embodiments, the polynucleotide encoding the light chain variable region has a nucleic acid sequence selected from SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:102. In some embodiments, the polynucleotide encoding the heavy chain variable region has a nucleic acid sequence selected from SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 SEQ ID NO:101 and SEQ ID NO:107.

In a related aspect, the invention further provides compositions comprising an antibody, antibody fragment, or antigen binding molecule of the invention, as described herein, and a pharmaceutically acceptable carrier. In some embodiments, the invention provides pharmaceutical compositions comprising an antibody, antibody fragment, or antigen binding molecule of the invention for administering to an individual.

In some embodiments, the composition further comprises a target antigen, for example, a cancer-associated antigen or a tumor-associated antigen. In some embodiments, the target antigen is a viral antigen, a bacterial antigen, a fungal antigen or a parasitic antigen.

In some embodiments, the composition further comprises an antagonist of CTLA4. In some embodiments, the composition further comprises an antagonist of LAG3. In some embodiments, the composition further comprises an antagonist of TIM3. In some embodiments, the composition further comprises an inhibitor of PD-1/PD-L1 (e.g., B7-H1 or analogue thereof, PD-1 antibody) interaction. In certain embodiments the composition further comprises an antagonist of PD-1. In certain embodiments the composition further comprises an antagonist of PD-L1.

In a further aspect, the invention further provides kits comprising an antibody or antibody fragment of the invention, as described herein.

In some embodiments, kits further comprise a second agent for co-administration with the antibody. In some embodiments, the second agent is a target antigen, for example, a cancer-associated antigen or a tumor-associated antigen. In some embodiments, the target antigen is a viral antigen, a bacterial antigen, a fungal antigen or a parasitic antigen.

In some embodiments, the second agent is an antagonist of CTLA4. In some embodiments, the second agent is an antagonist of TIM3. In some embodiments, the second agent is an antagonist of LAG3. In some embodiments, the second agent is an inhibitor of PD-1/PD-L1 (e.g., B7-H1 or analogue thereof, PD-1 antibody) interaction. In certain embodiments the second agent is an antagonist of PD-1. In certain embodiments the second agent is an antagonist of PD-L1.

Optionally, the antibody or antibody fragment and second agent are provided as a mixture. Optionally, the antibody or antibody fragment and the second agent are provided in separate formulations.

In another aspect, the invention provides methods of enhancing a T cell response in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an anti-GITR agonist antibody or antibody fragment of the invention, as described herein. In a further aspect, the invention provides an anti-GITR agonist antibody or antibody fragment of the invention for use in enhancing a T cell response in an individual. In a further aspect, the invention provides a composition comprising an antibody or antibody fragment of the invention for use in enhancing a T cell response in an individual.

In a further aspect, the invention provides methods of treating tumor growth of a cancer that expresses a tumor associated antigen in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an anti-GITR agonist antibody, antibody fragment, or antigen binding molecule of the invention, as described herein. The invention further provides an anti-GITR agonist antibody or antibody fragment of the invention for use in treating tumor growth of a cancer in an individual. The invention further provides a composition comprising an antibody or antibody fragment of the invention for use in reducing, inhibiting or preventing tumor growth of a cancer that expresses a tumor associated antigen in an individual.

With respect to embodiments of the methods and medical uses, in some embodiments, the anti-GITR agonist antibody, antibody fragment, or antigen binding molecule is co-administered with an antigen. In some embodiments, the antigen is a cancer-associated antigen or a tumor-associated antigen. In some embodiments, the anti-GITR agonist antibody or antibody fragment is co-administered with cancer cells from the patient, i.e., autologous cancer cells.

In some embodiments, the anti-GITR agonist antibody, antibody fragment, or antigen binding molecule is co-administered with an antagonist of CTLA4. In some embodiments, the anti-GITR agonist antibody, antibody fragment, or antigen binding molecule is co-administered with an antagonist of LAGS. In some embodiments, the anti-GITR agonist antibody, antibody fragment, or antigen binding molecule is co-administered with an antagonist of TIM3. In some embodiments, the anti-GITR agonist antibody or antibody fragment is co administered with an inhibitor of PD-1/PD-L1 (e.g., B7-H1) interaction. In certain embodiments, the anti-GITR agonist antibody, antibody fragment, or antigen binding molecule is co-administered with an antagonist of PD-1. In certain embodiments, the anti-GITR agonist antibody, antibody fragment, or antigen binding molecule is co-administered with an antagonist of PD-L1.

In some embodiments, the anti-GITR agonist antibody, antibody fragment, or antigen binding molecule is co-administered with a chemotherapeutic agent or a cytotoxin.

In some embodiments, the T cell response is a CD8+ cytotoxic T lymphocyte (CTL) T cell response. In some embodiments, the T cell response is a CD4+ helper T cell (Th) response.

In some embodiments provided compositions (e.g., anti-GITR agonist antibody, antibody fragment, or antigen binding molecules and methods of using them) can be used in combination with other agents or therapeutic modalities, e.g., a second therapeutic agent chosen from one or more of the agents listed in Table 6. In one embodiment, the methods described herein include administering to the subject an anti-GITR agonist antibody, antibody fragment, or antigen binding molecule as described herein (optionally in combination with one or more inhibitors of PD-1, PD-L1, LAG-3, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), or CTLA-4)), further include administration of a second therapeutic agent chosen from one or more of the agents listed in Table 6, in an amount effective to treat or prevent a disorder, e.g., a disorder as described herein, e.g., a cancer. When administered in combination, the—anti-GITR agonist antibody, antibody fragment, or antigen binding molecule, the additional agent (e.g., second or third agent), or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of the anti-GITR agonist antibody, antibody fragment, or antigen binding molecule, the additional agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy. In other embodiments, the amount or dosage of the anti-GITR agonist antibody, antibody fragment, or antigen binding molecule, the additional agent (e.g., second or third agent), or all, that results in a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower).

In other embodiments, the second therapeutic agent is chosen from one or more of the agents listed in Table 6 In one embodiment, the cancer is chosen from a lung cancer (e.g., a non-small cell lung cancer (NSCLC) (e.g., a NSCLC with squamous and/or non-squamous histology, or a NSCLC adenocarcinoma), or disclosed in a publication listed in Table 6. In some embodiments, the second therapeutic agent is chosen from one or more of: 1) a protein kinase C (PKC) inhibitor; 2) a heat shock protein 90 (HSP90) inhibitor; 3) an inhibitor of a phosphoinositide 3-kinase (PI3K) and/or target of rapamycin (mTOR); 4) an inhibitor of cytochrome P450 (e.g., a CYP17 inhibitor or a 17alpha-Hydroxylase/C17-20 Lyase inhibitor); 5) an iron chelating agent; 6) an aromatase inhibitor; 7) an inhibitor of p53, e.g., an inhibitor of a p53/Mdm2 interaction; 8) an apoptosis inducer; 9) an angiogenesis inhibitor; 10) an aldosterone synthase inhibitor; 11) a smoothened (SMO) receptor inhibitor; 12) a prolactin receptor (PRLR) inhibitor; 13) a Wnt signaling inhibitor; 14) a CDK4/6 inhibitor; 15) a fibroblast growth factor receptor 2 (FGFR2)/fibroblast growth factor receptor 4 (FGFR4) inhibitor; 16) an inhibitor of macrophage colony-stimulating factor (M-CSF); 17) an inhibitor of one or more of c-KIT, histamine release, Flt3 (e.g., FLK2/STK1) or PKC; 18) an inhibitor of one or more of VEGFR-2 (e.g., FLK-1/KDR), PDGFRbeta, c-KIT or Raf kinase C; 19) a somatostatin agonist and/or a growth hormone release inhibitor; 20) an anaplastic lymphoma kinase (ALK) inhibitor; 21) an insulin-like growth factor 1 receptor (IGF-1R) inhibitor; 22) a P-Glycoprotein 1 inhibitor; 23) a vascular endothelial growth factor receptor (VEGFR) inhibitor; 24) a BCR-ABL kinase inhibitor; 25) an FGFR inhibitor; 26) an inhibitor of CYP11B2; 27) a HDM2 inhibitor, e.g., an inhibitor of the HDM2-p53 interaction; 28) an inhibitor of a tyrosine kinase; 29) an inhibitor of c-MET; 30) an inhibitor of JAK; 31) an inhibitor of DAC; 32) an inhibitor of 11β-hydroxylase; 33) an inhibitor of IAP; 34) an inhibitor of PIM kinase; 35) an inhibitor of Porcupine; 36) an inhibitor of BRAF, e.g., BRAF V600E or wild-type BRAF; 37) an inhibitor of HER3; 38) an inhibitor of MEK; or 39) an inhibitor of a lipid kinase, e.g., as described herein and in Table 6.

Alternatively, or in combination with, the methods disclosed herein, the invention features a method of treating (e.g., inhibiting, reducing, ameliorating, or preventing) a disorder, e.g., a hyperproliferative condition or disorder (e.g., a cancer) in a subject. Methods include administering to the subject a combination of two, three or more therapeutic agents chosen from one, two or all of the following categories (i)-(iii): (i) an agent that enhances antigen (e.g., tumor antigen) presentation; (ii) an agent that enhances an effector cell response (e.g., B cell and/or T cell activation and/or mobilization); or (iii) an agent that decreases tumor immunosuppression, thereby treating the disorder, e.g., the hyperproliferative condition or disorder (e.g., the cancer). Exemplary therapeutic agents of categories (i)-(iii) are chosen from one or more of the agents listed in Table 7 and further described herein. In some embodiments, the combination includes at least a GITR modulator (e.g., an nti-GITR agonist antibody, antibody fragment, or antigen binding molecule as described herein) and one or more additional therapeutic agents of categories (i)-(iii). The cancer treated can be, e.g., a cancer described herein, such as lung cancer (squamous), lung cancer (adenocarcinoma), head and neck cancer, cervical cancer (squamous), stomach cancer, thyroid cancer, melanoma, nasopharyngeal cancer, or breast cancer.

In some embodiments, the patient has a cancer that expresses a tumor associated antigen. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, colorectal cancer, prostate, non-small cell lung cancer (NSCLC) and breast cancer. In one embodiment, the type of cancer is selected from the group consisting of: pancreatic cancer, melanomas, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, head and neck squamous cell carcinoma (HNSCC), liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and cancer of hematological tissues.

In some embodiments, the patient has an infectious disease, for example, a viral infection, a bacterial infection, a fungal antigen or a parasitic antigen. In some embodiments, the anti-GITR agonist antibody is co-administered with a viral antigen (e.g., from HCV, HSV or HIV). In some embodiments, the anti-GITR agonist antibody is co-administered with a bacterial antigen. In some embodiments, the anti-GITR agonist antibody is co-administered with a fungal antigen. In some embodiments, the anti-GITR agonist antibody is co-administered with a parasitic antigen (e.g., filariasis).

In still other embodiments, provided is an isolated antibody, antibody fragment, or antigen binding molecule for use in for use in therapy. In certain embodiments the antibody, antibody fragment or antigen binding molecule are provided for use enhancing a T cell response in an individual in need thereof. In certain embodiments the antibody, antibody fragment or antigen binding molecule are provided for use in the treatment of tumor growth in an individual in need thereof.

Provided combinations disclosed herein can result in one or more of: an increase in antigen presentation, an increase in effector cell function (e.g., one or more of T cell proliferation, IFN-γ secretion or cytolytic function), inhibition of regulatory T cell function, an effect on the activity of multiple cell types, such as regulatory T cell, effector T cells and NK cells), an increase in tumor infiltrating lymphocytes, an increase in T-cell receptor mediated proliferation, and a decrease in immune evasion by cancerous cells. Thus, such combinations can be used to treat or prevent disorders where enhancing an immune response in a subject is desired.

Accordingly, in another aspect, a method of modulating an immune response in a subject is provided. The method comprises administering to the subject a combination disclosed herein (e.g., a combination comprising a therapeutically effective amount of an anti-GITR antibody molecule), alone or in combination with one or more agents or procedures, such that the immune response in the subject is modulated. In one embodiment, the antibody molecule enhances, stimulates or increases the immune response in the subject. The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having, or at risk of having, a disorder described herein). In one embodiment, the subject is in need of enhancing an immune response. In one embodiment, the subject has, or is at risk of, having a disorder described herein, e.g., a cancer or an infectious disorder as described herein. In certain embodiments, the subject is, or is at risk of being, immunocompromised. For example, the subject is undergoing or has undergone a chemotherapeutic treatment and/or radiation therapy. Alternatively, or in combination, the subject is, or is at risk of being, immunocompromised as a result of an infection.

Definitions

An “antibody” refers to a polypeptide of the immunoglobulin family that is capable of noncovalently, reversibly, and specifically binding a corresponding antigen. An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” chain (about 25 kD) and one “heavy” chain (about 50-70 kD), connected through a disulfide bond. Recognized immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Antibodies of the invention can be of any isotype/class (e.g., IgG, IgM, IgA, IgD, and IgE), or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_(L)) and variable heavy chain (V_(H)) refer to these regions of light and heavy chains respectively. In addition to V regions, both heavy chains and light chains contain a constant (C) region or domain. A secreted form of a immunoglobulin C region is made up of three C domains, CH1, CH2, CH3, optionally CH4 (Cμ), and a hinge region. A membrane-bound form of an immunglobulin C region also has membrane and intracellular domains. Each light chain has a VL at the N-terminus followed by a constant domain (C) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain. The pairing of a VH and VL together forms a single antigen-binding site. A “conventional antibody” IgG immunoglobulin as used herein refers to an antibody in a configuration that occurs in nature. Typically, a conventional antibody IgG has four chains, two identical heavy chains and two identical light chains linked together through disulfide bonds. As used herein, an “antibody” also encompasses variations of antibodies and conventional antibody structures that possess a particular binding specificity, i.e., for GITR. Thus, within the scope of this concept are full length antibodies, chimeric antibodies, and humanized antibodies, that possess a particular binding specificity for GITR.

Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′₂, a dimer of Fab′ which itself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. The F(ab)′₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region. Paul, Fundamental Immunology 3d ed. (1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. As used herein, an “antibody fragment” refers to one or more portions of an antibody, either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies, that retain binding specificity and agonist activity for GITR. Examples of antibody fragments include Fv fragments, single chain antibodies (ScFv), Fab, Fab′, Fd (Vh and CH1 domains), dAb (Vh and an isolated CDR); and multimeric versions of these fragments (e.g., F(ab′)₂,) with the same binding specificity. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, diabodies, triabodies, tetrabodies, vNAR, bis-scFv, and other variations of antibody-like compounds to achieve the binding specificity and activity provided in the present invention.

A “Fab” domain as used in the context of the invention comprises a heavy chain variable domain, a constant region CH1 domain, a light chain variable domain, and a light chain constant region CL domain. The interaction of the domains is stabilized by a disulfide bond between the CH1 and CL domains. In some embodiments, the heavy chain domains of the Fab are in the order, from N-terminus to C-terminus, VH-CH and the light chain domains of a Fab are in the order, from N-terminus to C-terminus, VL-CL. In some embodiments, the heavy chain domains of the Fab are in the order, from N-terminus to C-terminus, CH—VH and the light chain domains of the Fab are in the order CL-VL. Although Fabs were historically identified by papain digestion of an intact immunoglobulin, in the context of this invention, a “Fab” is typically produced recombinantly by any method. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

The C-terminal portion of the immunoglobulin heavy chains, comprising the CH2 and CH3 domains, is the “Fc” domain. An “Fc region” as used herein refers to the constant region of an antibody excluding the first constant region immunoglobulin domain. Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ. It is understood in the art that the boundaries of the Fc region may vary, however, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, using the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). “Fc region” may refer to this region in isolation or this region in the context of an antibody or antibody fragment. “Fc region” includes naturally occurring allelic variants of the Fc region, e.g., in the CH2 and CH3 region, as well as modifications that modulate effector function. Fc regions also include variants that don't result in alterations to biological function. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. For example, in certain embodiments a C-terminal lysine may be modified replaced or removed. In particular embodiments one or more C-terminal residues in the Fc region is altered or removed. In certain embodiments one or more C-terminal residues in the Fc (e.g., the terminal lysine) is deleted. In certain other embodiments one or more C-terminal residues in the Fc is substituted with an alternate amino acid (e.g., the terminal lysine is replaced). Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, et al., Science 247:306-1310, 1990). The Fc domain is the portion of the Ig recognized by cell receptors, such as the FcR, and to which the complement-activating protein, C1 q, binds. The lower hinge region, which is encoded in the 5′ portion of the CH2 exon, provides flexibility within the antibody for binding to FcR receptors.

“Complementarity-determining domains” or “complementary-determining regions (“CDRs”) interchangeably refer to the hypervariable regions of V_(L) and V_(H). CDRs are the target protein-binding site of the antibody chains that harbors specificity for such target protein. There are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in each human V_(L) or V_(H), constituting about 15-20% of the variable domains. CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for the binding specificity. The remaining stretches of the V_(L) or V_(H), the so-called framework regions, exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).

Positions of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT) (on the worldwide web at imgt.cines.fr/), and AbM (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). Definitions of antigen combining sites are also described in the following: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M. P., Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg M. J. E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

Under Kabat, CDR amino acid residues in the V_(H) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the V_(L) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, CDR amino acids in the V_(H) are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in V_(L) are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.

The term “binding specificity determinant” or “BSD” interchangeably refer to a minimum contiguous or non-contiguous amino acid sequence within a complementary determining region necessary for determining the binding specificity of an antibody. A minimum binding specificity determinant can be within one or more CDR sequences. In some embodiments, the minimum binding specificity determinants reside within (i.e., are determined solely by) a portion or the full-length of the CDR3 sequences of the heavy and light chains of the antibody.

An “antibody light chain” or an “antibody heavy chain” as used herein refers to a polypeptide comprising the V_(L) or V_(H), respectively. The endogenous V_(L) is encoded by the gene segments V (variable) and J (junctional), and the endogenous V_(H) by V, D (diversity), and J. Each of V_(L) or V_(H) includes the CDRs as well as the framework regions. In this application, antibody light chains and/or antibody heavy chains may, from time to time, be collectively referred to as “antibody chains.” These terms encompass antibody chains containing mutations that do not disrupt the basic structure of V_(L) or V_(H), as one skilled in the art will readily recognize.

The term “valency” as used herein refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site may specifically bind the same or different molecules (e.g., may bind to different molecules, e.g., different antigens, or different epitopes on the same molecule). A conventional antibody, for example, has two binding sites and is bivalent. The antibodies, antigen binding molecules, and fragments thereof, can be monovalent (i.e., bind one target molecule), bivalent, or multivalent (i.e., bind more than one target molecule).

For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96. Alan R. Liss, Inc. 1985). Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express primatized or humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., supra; Marks et al., Biotechnology, 10:779-783, (1992)).

Methods for primatizing or humanizing non-human antibodies are well known in the art. Generally, a primatized or humanized antibody has one or more amino acid residues introduced into it from a source which is non-primate or non-human. These non-primate or non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, primatized or humanized antibodies are typically primate or human antibodies in which some complementary determining region (“CDR”) residues and possibly some framework (“FR”) residues are substituted by residues from analogous sites in an originating species (e.g., rodent antibodies) to confer binding specificity.

A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, and drug; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

Antibodies or antigen-binding molecules of the invention further include one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins. It also includes bispecific antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Other antigen-binding fragments or antibody portions of the invention include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies.

The various antibodies or antigen-binding fragments described herein can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv), or identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990). For example, minibodies can be generated using methods described in the art, e.g., Vaughan and Sollazzo, Comb Chem High Throughput Screen. 4:417-30 2001. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Single chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffled libraries. Such libraries can be constructed from synthetic, semi-synthetic or native and immunocompetent sources.

The term “antigen binding molecule” or “non-antibody ligand” refers to antibody mimics that use non-immunoglobulin protein scaffolds, including but not limited to, adnectins, avimers, single chain polypeptide binding molecules, and antibody-like binding peptidomimetics.

The term “variable region” or “V-region” interchangeably refer to a heavy or light chain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An endogenous variable region is encoded by immunoglobulin heavy chain V-D-J genes or light chain V-J genes. A V-region can be naturally occurring, recombinant or synthetic.

As used herein, the term “variable segment” or “V-segment” interchangeably refer to a subsequence of the variable region including FR1-CDR1-FR2-CDR2-FR3. An endogenous V-segment is encoded by an immunoglobulin V-gene. A V-segment can be naturally occurring, recombinant or synthetic.

As used herein, the term “J-segment” refers to a subsequence of the variable region encoded comprising a C-terminal portion of a CDR3 and the FR4. An endogenous J-segment is encoded by an immunoglobulin J-gene. A J-segment can be naturally occurring, recombinant or synthetic.

A “humanized” antibody is an antibody that retains the reactivity (e.g., binding specificity, activity) of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining non-human CDR regions and replacing the remaining parts of the antibody with human counterparts. See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994).

The term “corresponding human germline sequence” refers to the nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. The corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be framework regions only, complementary determining regions only, framework and complementary determining regions, a variable segment (as defined above), or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods described herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence. Corresponding human germline sequences can be determined, for example, through the publicly available international ImMunoGeneTics database (IMGT) (on the worldwide web at imgt.cines.fr/) and V-base (on the worldwide web at vbase.mrc-cpe.cam.ac.uk).

The phrase “specifically binds” or “selectively binds,” when used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binding agent, refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologics, e.g., in a biological sample, e.g., a blood, serum, plasma or tissue sample. Thus, under certain designated immunoassay conditions, the antibodies or binding agents with a particular binding specificity bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample. In one embodiment, under designated immunoassay conditions, the antibody or binding agents with a particular binding specificity bind to a particular antigen at least ten (10) times the background and do not substantially bind in a significant amount to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to have been selected for its specificity for a particular protein (e.g., human GITR). As used herein, specific binding includes antibodies fragments thereof and binding molecules that selectively bind to human GITR and do not include antibodies that cross-react with, e.g., murine GITR molecules or other TNF receptor superfamily members. In some embodiments, antibodies or antibody fragments are selected that cross-react with non-human primate GITR (e.g., cynomolgus GITR).

A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective binding reaction will produce a signal at least twice over the background signal and more typically at least than 10 to 100 times over the background.

The term “equilibrium dissociation constant (K_(D), M)” refers to the dissociation rate constant (k_(d), time⁻¹) divided by the association rate constant (k_(a), time⁻¹, M⁻¹). Equilibrium dissociation constants can be measured using any known method in the art. The antibodies of the present invention generally will have an equilibrium dissociation constant of less than about 10⁻⁷ or 10⁻⁸ M, for example, less than about 10⁻⁹ M or 10⁻¹⁰ M, in some embodiments, less than about 10⁻¹¹ M, 10⁻¹² M or 10⁻¹³ M. In some embodiments, the isolated antibody or antibody fragment binds to human GITR with an equilibrium dissociation constant (K_(D)) of about 1 nM or less. In some embodiments, the antibody or antibody fragment binds to human GITR with a K_(D) that is less than 1 nM. In some embodiments, the antibody or antibody fragment binds to human GITR with a K_(D) that is in the range of from about 0.5 nM to about 1.0 nM.

As used herein, the term “antigen-binding region” refers to a domain of the GITR-binding molecule of this invention that is responsible for the specific binding between the molecule and GITR. An antigen-binding region includes at least one antibody heavy chain variable region and at least one antibody light chain variable region. There are at least one such antigen-binding regions present in each GITR-binding molecule of this invention, and each of the antigen-binding regions may be identical or different from the others. In some embodiments, at least one of the antigen-binding regions of a GITR-binding molecule of this invention acts as an agonist of GITR.

The term “antibody agonist” or “agonist” interchangeably refer to an antibody capable of activating a receptor to induce a full or partial receptor-mediated response. For example, an agonist of GITR binds to GITR and induces GITR-mediated intracellular signaling (e.g., increased NF-κB expression activation). The antibody agonist stimulates signaling through GITR similarly to the native ligand, GITR-L. Binding of GITR-L to GITR induces NFκB activation due to degradation of IκB. In some embodiments, a GITR antibody agonist can be identified by its ability to bind GITR and induce T cell (e.g., CD8⁺ CTLs or CD4⁺ Th cells) proliferation, survival, cytolytic activity and/or cytokine production (e.g., IFNγ, IL-10, IL-13, TNFα) or as otherwise described herein.

The term “GITR” or “glucocorticoid-induced tumor necrosis factor receptor receptor” or “tumor necrosis factor receptor superfamily, member 18” or “TNFRSF18” interchangeably refer to a type I transmembrane protein that is a member of the TNF-receptor superfamily. GITR is expressed at high levels on CD4⁺ CD25⁺ and on activated effector CD4⁺ and CD8⁺ T cells. The nucleic acid and amino acid sequences of GITR are known, and have been published in

GenBank Accession Nos. NM_004195.2 → NP_004186.1 (isoform 1 precursor), SEQ ID NO: 1: 1 maqhgamgaf ralcglallc alslgqrptg gpgcgpgrll lgtgtdarcc rvhttrocrd 61 ypgeeccsew dcmcvqpefh cgdpccttcr hhpcppgqgv qsqgkfsfgf qcidcasgtf 121 sggheghckp wtdctqfgfl tvfpgnkthn avcvpgsppa eplgwltvvl lavaacvlll 181 tsaqlglhiw qlrsqcmwpr etqlllevpp stedarscqf peeergersa eekgrlgdlw 241 v; NM_148901.1 → NP_683699.1 (isoform 2 precursor), SEQ ID NO: 2: 1 maqhgamgaf ralcglallc alslgqrptg gpgcgpgrll lgtgtdarcc rvhttrocrd 61 ypgeeccsew dcmcvqpefh cgdpccttcr hhpcppgqgv qsqgkfsfgf qcidcasgtf 121 sggheghckp wtdccwrcrr rpktpeaass prksgasdrq rrrggwetcg cepgrppgpp 181 taaspspgap qaagalrsal grallpwqqk wvqeggsdqr pgpcssaaaa gpcrreretq 241 swppsslagp dgvgs; and NM_148902.1 → NP_683700.1 (isoform 3 precursor), SEQ ID NO: 3: 1 maqhgamgaf ralcglallc alslgqrptg gpgcgpgrll lgtgtdarcc rvhttrccrd 61 ypgeeccsew dcmcvqpefh cgdpccttcr hhpcppgqgv qsqgkfsfgf qcidcasgtf 121 sggheghckp wtdctqfgfl tvfpgnkthn avcvpgsppa eplgwltvvl lavaacvlll 181 tsaqlglhiw qlrktqllle vppstedars cqfpeeerge rsaeekgrlg dlwv. See also, GenBank Accession No. NM_005092→NP_005083.2. Structurally, a GITR amino acid sequence is a type I transmembrane protein that is a member of the TNF-receptor superfamily having a signal peptide, an extracellular domain (ECD) comprising three cysteine-rich domains (CRDs) and has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of GenBank accession numbers NP_004186.1 (SEQ ID NO:1), NP_683699.1 (SEQ ID NO:2), NP_683700.1 (SEQ ID NO:3), or NP_005083.2. Structurally, a GITR nucleic acid sequence has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence of GenBank accession numbers NM_004195.2, NM_148901.1, NM_148902.1, NM_005092 or SEQ ID NOs:1-4. Functionally, agonism of rodent GITR inhibits, at least transiently, suppressor activity of CD25⁺ regulatory T cells (Treg). GITR agonism further enhances immunoactivity, e.g., proliferation, survival, cytokine production and cytolytic activity of activated effector CD4⁺ and CD8⁺ T cells. See, e.g., Nocentini, et al., Eur J Immunol (2007) 37:1165-1169; Expert Opin Ther Patents (2007) 17(5):567-757; Shevach and Stephens, Nature Reviews Immunology (2006) 6:613-618.

“Activity” of a polypeptide of the invention refers to structural, regulatory, or biochemical functions of a polypeptide in its native cell or tissue. Examples of activity of a polypeptide include both direct activities and indirect activities. Exemplary activities of GITR agonism include intracellular signaling that results in increased activation of NF-κB, increased proliferation, survival, cytokine production (e.g., IFNγ, IL-10, IL-13, TNFα), and cytolytic activity of activated effector CD4⁺ and CD8⁺ T cells. Therapeutically, agonism of GITR augments antitumor and antiviral T-cell responses in vivo.

The term “isolated,” when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. It can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The invention provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein (e.g., the variable regions exemplified in any one of SEQ ID NOS:6-10, 12, 14, 59 and 61; the variable segments exemplified in any one of SEQ ID NOS:16-17; the CDRs exemplified in any one of SEQ ID NOS:22-34; the FRs exemplified in any one of SEQ ID NOS:35-50; and the nucleic acid sequences exemplified in any on of SEQ ID NOS:51-58 and 60). Optionally, the identity exists over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

The term “link,” when used in the context of describing how the antigen-binding regions are connected within a GITR-binding molecule of this invention, encompasses all possible means for physically joining the regions. The multitude of antigen-binding regions are frequently joined by chemical bonds such as a covalent bond (e.g., a peptide bond or a disulfide bond) or a non-covalent bond, which can be either a direct bond (i.e., without a linker between two antigen-binding regions) or indirect bond (i.e., with the aid of at least one linker molecule between two or more antigen-binding regions).

The terms “subject,” “patient,” and “individual” interchangeably refer to a mammal, for example, a human or a non-human primate mammal. The mammal can also be a laboratory mammal, e.g., mouse, rat, rabbit, hamster. In some embodiments, the mammal can be an agricultural mammal (e.g., equine, ovine, bovine, porcine, camelid) or domestic mammal (e.g., canine, feline).

As used herein, the terms “treat,” “treating,” or “treatment” of any disease or disorder refer in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, “treat,” “treating,” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treat,” “treating,” or “treatment” refers to preventing or delaying the onset or development or progression of the disease or disorder.

The term “therapeutically acceptable amount” or “therapeutically effective dose” interchangeably refer to an amount sufficient to effect the desired result (i.e., a reduction in tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal or parasitic infection). In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved. A “prophylactically effective amount,” and a “therapeutically effective amount,” of a GITR agonizing antibody of the invention can prevent the onset of, or result in a decrease in severity of, respectively, disease symptoms, including symptoms associated with cancer or infectious disease. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the modified antibody or antibody fragment may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the modified antibody or antibody fragment is outweighed by the therapeutically beneficial effects. A “therapeutically effective dosage” preferably inhibits a measurable parameter, e.g., tumor growth rate by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit a measurable parameter, e.g., cancer, can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.

The term “co-administer” refers to the simultaneous presence of two active agents in the blood of an individual. Active agents that are co-administered can be concurrently or sequentially delivered.

As used herein, the phrase “consisting essentially of” refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any inactive carrier or excipients for the intended purpose of the methods or compositions. In some embodiments, the phrase “consisting essentially of” expressly excludes the inclusion of one or more additional active agents other than an agonist anti-GITR antibody of the invention. In some embodiments, the phrase “consisting essentially of” expressly excludes the inclusion of one or more additional active agents other than an agonist anti-GITR antibody of the invention and a second co-administered agent.

The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. The terms “tumor” and “cancer” are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.

The terms “cancer-associated antigen” or “tumor-associated antigen” or “tumor-specific marker” or “tumor marker” interchangeably refers to a molecule (typically protein, carbohydrate or lipid) that is preferentially expressed on the surface of a cancer cell in comparison to a normal cell, and which is useful for the preferential targeting of a pharmacological agent to the cancer cell. Oftentimes, a cancer-associated antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, 1-fold over expression, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell. Oftentimes, a cancer-associated antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. Oftentimes, a cancer-associated antigen will be expressed exclusively on the cell surface of a cancer cell and not synthesized or expressed on the surface of a normal cell. Exemplified cell surface tumor markers include the proteins c-erbB-2 and human epidermal growth factor receptor (HER) for breast cancer, PSMA for prostate cancer, and carbohydrate mucins in numerous cancers, including breast, ovarian and colorectal.

The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC's) on its surface. T-cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

The term “costimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include, but are not limited to an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

“Immune effector cell,” or “effector cell” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

“Immune effector” or “effector” “function” or “response,” as that term is used herein, refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. E.g., an immune effector function or response refers a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector function or response.

The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.

As used herein, the terms “first”, “second”, “third” and “fourth”, with respect to antigen binding moieties, e.g., Fabs, are used for convenience of distinguishing when there is more than one of each moiety. Use of these terms is not intended to confer a specific order or orientation of the antibody unless otherwise stated.

The terms “a,” “an,” and “the” include plural referents, unless the context clearly indicates otherwise

Agonist Anti-GITR Antibodies

The present invention provides antibodies, antibody fragments, and antigen binding molecules that bind to and stimulate signaling through GITR and/or induce a potentiated immune response in vivo. The antibodies, antibody fragments, and antigen binding molecules find uses in enhancing CD4+ T helper (Th) and/or CD8+ cytolytic T lymphocyte (CTL) responses against a target antigen. They also find uses in treating disease conditions whose progression can be reversed or inhibited by an effective immune response, including cancers and infectious diseases.

The antibodies, antibody fragments and antigen binding molecules of the present invention show suitable properties to be used in human patients, for example, they have low risk for immunogenicity issues for uses in human (they are encoded by human germline nucleic acid sequences, with the exception of the binding specificity determining regions (BSD), in particular at least CDR3); have high affinity to GITR (e.g., K_(D) is at least less than 5 nM); do not cross-react with other members of the TNFR superfamily; cross-react with human and non-human primate GITR; and agonize GITR signaling at low doses (e.g., in concentrations of less than 5 nM in in vitro assays). Other activities and characteristics are also demonstrated throughout the specification.

Accordingly, the present invention provides antibodies, antibody fragments, and antigen-binding molecules that are agonists of GITR. Provided anti-GITR antibodies, antibody fragments, or antigen-binding molecules contain a minimum binding sequence determinant (BSD) within the CDR3 of the heavy and light chains derived from the originating or reference monoclonal antibody, for example, the antibodies described in Table 1 and Table 2 below. The remaining sequences of the heavy chain and light chain variable regions (CDR and FR), e.g., V-segment and J-segment, are from corresponding human germline and affinity matured amino acid sequences. The V-segments can be selected from a human V-segment library. Further sequence refinement can be accomplished by affinity maturation or other methods known in the art to optimize binding activity or activity of the antibodies, antibody fragments or antigen binding molecules of the invention.

In another embodiment, heavy and light chains of the anti-GITR antibodies or antibody fragments contain a human V-segment from the corresponding human germline sequence (FR1-CDR1-FR2-CDR2-FR3), e.g., selected from a human V-segment library, and a CDR3-FR4 sequence segment from the originating monoclonal antibody (e.g., the antibodies as described in Table 1 and Table 2). The CDR3-FR4 sequence segment can be further refined by replacing sequence segments with corresponding human germline sequences and/or by affinity maturation. For example, the FR4 and/or the CDR3 sequence surrounding the BSD can be replaced with the corresponding human germline sequence, while the BSD from the CDR3 of the originating monoclonal antibody is retained.

In some embodiments, the corresponding human germline sequence for the heavy chain V-segment is VH3 3-13/30: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIRYDGSN KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO:89). In one embodiment, the last amino acid in SEQ ID NO:89, lysine (“K”), is substituted with arginine (“R”). In some embodiments, the corresponding human germline sequence for the heavy chain J-segment is JH4. In some embodiments, the heavy chain J-segment comprises the human germline JH4 partial sequence WGQGTLVTVSS (SEQ ID NO:90). The full-length J-segment from human germline JH4 is YFDYWGQGTLVTVSS (SEQ ID NO:91). The variable region genes are referenced in accordance with the standard nomenclature for immunoglobulin variable region genes. Current immunoglobulin gene information is available through the worldwide web, for example, on the ImMunoGeneTics (IMGT), V-base and PubMed databases. See also, Lefranc, Exp Clin Immunogenet. 2001; 18(2):100-16; Lefranc, Exp Clin Immunogenet. 2001; 18(3):161-74; Exp Clin Immunogenet. 2001; 18(4):242-54; and Giudicelli, et al., Nucleic Acids Res. 2005 Jan. 1; 33(Database issue):D256-61.

In some embodiments, the corresponding human germline sequence for the light chain V-segment is VKIII L16/A27: EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP (SEQ ID NO:92). In some embodiments, the corresponding human germline sequence for the light chain J-segment is JK2. In some embodiments, the light chain J-segment comprises the human germline Jk2 partial sequence FGQGTKLEIK (SEQ ID NO:93). The full-length J segment from human germline Jk2 is YTFGQGTKLEIK (SEQ ID NO:94).

In some embodiments, the heavy chain V-segment has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence

(SEQ ID NO: 16) (E/Q)VQLVESGGGLVQ(P/S)GGSLRLSCAASGFSLSSYGVDWVRQA PGKGLEW(L/V)GVIWGGGGTYY(A/T)(A/S)S(L/V)M(A/G)RF TISRDNSKNTLYLQMNSLRAEDTAVYYCA(K/R)(H/N)AYGHDGGFAM DYWGQGTLVTVSS.

In some embodiments, the light chain V-segment has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence

(SEQ ID NO: 17) EIVMTQSPATLSVSPGERATLSCRAS(E/Q)SVSSN(L/V)AWYQQ (K/R)PGQAPRLLIYGASNRATGIP(D/A)RFSGSGSGTDFTLTIS RLEPEDFAVYYCGQSYSYPFTFGQGTKLEIK.

In some embodiments, i) the heavy chain CDR3 comprises the amino acid sequence HAYGHDGGFAMDY (SEQ ID NO:29) or NAYGHDGGFAMDY (SEQ ID NO:109); and ii) the light chain CDR3 variable region comprises the amino acid sequence GQSYSYPFT (SEQ ID NO:34), or SYSYPF (SEQ ID NO:83).

In some embodiments, the antibodies or antibody fragments of the invention comprise a heavy chain variable region comprising a CDR1 comprising an amino acid sequence SYGVD (SEQ ID NO:22), or GFSLSSY (SEQ ID NO:84); a CDR2 comprising an amino acid sequence VIWGGGGTYY(A/T)(A/S)S(L/V)M(A/G) (SEQ ID NO:28), or WGGGG (SEQ ID NO:80); and a CDR3 comprising an amino acid sequence of HAYGHDGGFAMDY (SEQ ID NO:29) or NAYGHDGGFAMDY (SEQ ID NO:109).

In some embodiments, the antibodies or antibody fragments of the invention comprise a light chain variable region comprising a CDR1 comprising an amino acid sequence RAS(E/Q)SVSSN(L/V)A (SEQ ID NO:32) or S(E/Q)SVSSN (SEQ ID NO:87); a CDR2 comprising an amino acid sequence GASNRAT (SEQ ID NO:33), or GAS (SEQ ID NO:82); and a CDR3 comprising an amino acid sequence of GQSYSYPFT (SEQ ID NO:34), or SYSYPF (SEQ ID NO:83).

In some embodiments, the antibodies or antibody fragments of the invention comprise a heavy chain variable region comprising a CDR1 comprising an amino acid sequence SYGVD (SEQ ID NO:22), or GFSLSSY (SEQ ID NO:84); a CDR2 comprising an amino acid sequence VIWGGGGTYY(A/T)(A/S)S(L/V)M(A/G) (SEQ ID NO:28) or WGGGG (SEQ ID NO:80); and a CDR3 comprising an amino acid sequence of HAYGHDGGFAMDY (SEQ ID NO:29) or NAYGHDGGFAMDY (SEQ ID NO:109). Such antibodies or antibody fragments of the invention further comprise a light chain variable region comprising a CDR1 comprising an amino acid sequence RAS(E/Q)SVSSN(L/V)A (SEQ ID NO:32), or S(E/Q)SVSSN (SEQ ID NO:87); a CDR2 comprising an amino acid sequence GASNRAT (SEQ ID NO:33), or GAS (SEQ ID NO:82); and a CDR3 comprising an amino acid sequence of GQSYSYPFT (SEQ ID NO:34), or SYSYPF (SEQ ID NO:83).

In some embodiments, the antibodies or antibody fragments of the invention comprise a heavy chain variable region comprising a CDR1 comprising an amino acid sequence SYGVD (SEQ ID NO:22), or GFSLRSY (SEQ ID NO:79); a CDR2 comprising an amino acid sequence VIWGGGGTNYNSALMA (SEQ ID NO:62), or WGGGG (SEQ ID NO:80); and a CDR3 comprising an amino acid sequence of HAYGHDGGFAMDY (SEQ ID NO:29) or NAYGHDGGFAMDY (SEQ ID NO:109). In some embodiments, the antibodies or antibody fragments are humanized.

In some embodiments, the antibodies or antibody fragments of the invention comprise a light chain variable region comprising a CDR1 comprising an amino acid sequence KASENVDTFVS (SEQ ID NO:63), or SENVDTF (SEQ ID NO:81); a CDR2 comprising an amino acid sequence GASNRYT (SEQ ID NO:64), or GAS (SEQ ID NO:82); and a CDR3 comprising an amino acid sequence of GQSYSYPFT (SEQ ID NO:34), or SYSYPF (SEQ ID NO:83). In some embodiments, the antibodies or antibody fragments are humanized.

In some embodiments, the antibodies or antibody fragments of the invention comprise a heavy chain variable region comprising a CDR1 comprising an amino acid sequence SYGVD (SEQ ID NO:22), or GFSLRSY (SEQ ID NO:79); a CDR2 comprising an amino acid sequence VIWGGGGTNYNSALMA (SEQ ID NO:62), or WGGGG (SEQ ID NO:80); and a CDR3 comprising an amino acid sequence of HAYGHDGGFAMDY (SEQ ID NO:29) or NAYGHDGGFAMDY (SEQ ID NO:109). Such antibodies or antibody fragments further comprise a light chain variable region comprising a CDR1 comprising an amino acid sequence KASENVDTFVS (SEQ ID NO:63), or SENVDTF (SEQ ID NO:81); a CDR2 comprising an amino acid sequence GASNRYT (SEQ ID NO:64), or GAS (SEQ ID NO:82); and a CDR3 comprising an amino acid sequence of GQSYSYPFT (SEQ ID NO:34), or SYSYPF (SEQ ID NO:83). In some embodiments, the antibodies or antibody fragments are humanized.

In some embodiments, the heavy chain variable region comprises a FR1 comprising the amino acid sequence of (E/Q)VQLVESGGGLVQ(P/S)GGSLRLSCAASGFSLS (SEQ ID NO:37); a FR2 comprising the amino acid sequence of WVRQAPGKGLEW(L/V)G (SEQ ID NO:40); a FR3 comprising the amino acid sequence of RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(K/R) (SEQ ID NO:41); and a FR4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO:42). In some embodiments, the heavy chain variable region comprises a FR1 comprising the amino acid sequence selected from QVQLVESGGGLVQPGGSLRLSCAASGFSLS (SEQ ID NO:35) and QVQLVESGGGLVQPGGSLRLSCAASGFSLS (SEQ ID NO:36); a FR2 comprising the amino acid sequence selected from WVRQAPGKGLEWVG (SEQ ID NO:38) and WVRQAPGKGLEWLG (SEQ ID NO:39); a FR3 comprising the amino acid sequence of SEQ ID NO:41; and a FR4 comprising the amino acid sequence of SEQ ID NO:42. The identified amino acid sequences may have one or more substituted amino acids (e.g., from affinity maturation) or one or two conservatively substituted amino acids.

In some embodiments, the light chain variable region comprises a FR1 comprising an amino acid sequence of EIVMTQSPATLSVSPGERATLSC (SEQ ID NO:43); a FR2 comprising the amino acid sequence of WYQQ(K/R)PGQAPRLLIY (SEQ ID NO:46); a FR3 comprising the amino acid sequence of GIP(A/D)RFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:49); and a FR4 comprising the amino acid sequence of SEQ ID NO:50. In some embodiments, the light chain variable region comprises a FR1 comprising an amino acid sequence of SEQ ID NO:43; a FR2 comprising the amino acid sequence selected from WYQQRPGQAPRLLIY (SEQ ID NO:44) and WYQQKPGQAPRLLIY (SEQ ID NO:45); a FR3 comprising the amino acid sequence selected from GIPARFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:47) and GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:48); and a FR4 comprising the amino acid sequence of FGQGTKLEIK (SEQ ID NO:50). The identified amino acid sequences may have one or more substituted amino acids (e.g., from affinity maturation) or one or two conservatively substituted amino acids.

Over their full length, the variable regions of the anti-GITR antibodies of the present invention generally will have an overall variable region (e.g., FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) amino acid sequence identity of at least about 85%, for example, at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to the corresponding human germline variable region amino acid sequence. For example, the heavy chain of the anti-GITR antibodies can have at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the human germline variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIRYDGSN KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK-YFDYWGQGTLVTVSS (SEQ ID NOS:89 and 91)(VH3 3-13/30+CDR3+JH4, the hyphen represents CDR3, which may be variable in length). In one embodiment, the last amino acid in SEQ ID NO:89, lysine (K), is substituted with arginine (R). The light chain of the anti-GITR antibodies can have at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the human germline variable region EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYC-YTFGQGTKLEIK (SEQ ID NOS:98 and 94) (VKIII L16/A27+CDR3+JK2; the hyphen represents CDR3, which may be variable in length). In some embodiments, only amino acids within the framework regions are added, deleted, or substituted. In some embodiments, the sequence identity comparison excludes the CDR3.

TABLE 1 Examples of anti-GITR agonist antibodies of the present invention. SEQ ID NO: Amino acid or polynucleotide (PN) description Sequence 61: VH, MAB1 QVQLKESGPGLVAPSQSLSITCTVSGFSLRSYGVDWVRQPPGKGLEWLGVIWGG GGTNYNSALMAKLSISKDKSKSQVFLKMNSLQTDDTAMYYCAKHAYGHDGGFAM DYWGQGTSVTVSS 59: VL, MAB1 NIVMTQSPKSMSMSVGERVTLSCKASENVDTFVSWYQQKPDHSPKLLIYGASNR YTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYSYPFTFGSGTKLEIK 60: PN of VH, CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTG MAB1 TCCATCACTTGCACTGTCTCTGGGTTTTCATTAAGGAGCTATGGTGTAGACTGG encoding GTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGT SEQ ID NO: 61 GGAGGCACAAATTATAATTCAGCTCTCATGGCCAAACTGAGTATCAGCAAAGAC (VH) AAGTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACA GCCATGTACTACTGTGCCAAACATGCCTATGGTCACGACGGCGGTTTTGCTATG GACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 58: PN of VL, AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGG MAB1 GTCACCTTGAGCTGCAAGGCCAGTGAGAATGTGGATACTTTTGTATCCTGGTAT encoding CAACAGAAACCAGACCACTCTCCTAAACTACTGATATACGGGGCATCCAACCGG SEQ ID NO: 59 TACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACT (VL) CTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAG AGTTACAGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA 6: VH, MAB2 QVQLVESGGGLVQPGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWVGVIWGG GGTYYASSVMARFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHAYGHDGGFAM DYWGQGTLVTVSS 7: VL, MAB2 EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIK 65: Heavy QVQLVESGGGLVQPGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWVGVIWGG chain, MAB2 GGTYYASSVMARFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHAYGHDGGFAM DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 66: Light chain, EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR MAB2 ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 51: PN of VH, CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG MAB2 AGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGCAGCTATGGTGTGGACTGG encoding GTTCGCCAGGCTCCAGGAAAGGGTCTGGAGTGGGTGGGAGTTATATGGGGTGGT SEQ ID NO: 6 GGAGGCACATATTATGCTTCTTCTGTCATGGCCAGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG GCCGTGTATTACTGCGCCAAACATGCCTATGGCCATGATGGCGGCTTTGCTATG GATTATTGGGGCCAGGGTACCCTTGTGACCGTGAGCTCA 52: PN of VL, GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCTCCAGGAGAAAGA MAB2 GCCACCCTCTCCTGCAGGGCCAGTGAGAGTGTTAGCAGTAATGTAGCCTGGTAC encoding CAGCAGAGACCTGGCCAGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGG SEQ ID NO: 7 GCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGCGGCCAG AGCTATAGCTATCCATTTACCTTTGGCCAGGGCACCAAGCTTGAAATTAAG 67: PN of HC, CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG MAB2 AGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGCAGCTATGGTGTGGACTGG encoding GTTCGCCAGGCTCCAGGAAAGGGTCTGGAGTGGGTGGGAGTTATATGGGGTGGT SEQ ID NO: 65 GGAGGCACATATTATGCTTCTTCTGTCATGGCCAGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG GCCGTGTATTACTGCGCCAAACATGCCTATGGCCATGATGGCGGCTTTGCTATG GATTATTGGGGCCAGGGTACCCTTGTGACCGTGAGCTCAGCTAGCACCAAGGGC CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCC GCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGG AACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGC ACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCA GCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG AGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG CACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTG GTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAG TGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAG GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCC AGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTG ACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG 68: PN of LC, GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCTCCAGGAGAAAGA MAB2 GCCACCCTCTCCTGCAGGGCCAGTGAGAGTGTTAGCAGTAATGTAGCCTGGTAC encoding CAGCAGAGACCTGGCCAGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGG SEQ ID NO: 66 GCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGCGGCCAG AGCTATAGCTATCCATTTACCTTTGGCCAGGGCACCAAGCTTGAAATTAAGCGT ACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCC AAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGC GTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACC CTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCAC CAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 8: VH, MAB3 QVQLVESGGGLVQPGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWLGVIWGG GGTYYTASLMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHAYGHDGGFAM DYWGQGTLVTVSS 9: VL, MAB3 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASNR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIK 69: Heavy QVQLVESGGGLVQPGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWLGVIWGG chain, MAB3 GGTYYTASLMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHAYGHDGGFAM DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 70: Light chain, EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASNR MAB3 ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 53: PN of VH, CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG MAB3 AGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGCAGCTATGGTGTGGACTGG encoding GTTCGCCAGGCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGT SEQ ID NO: 8 GGAGGCACATATTATACTGCTTCTCTCATGGGCAGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG GCCGTGTATTACTGCGCCAAACATGCCTATGGCCATGATGGCGGCTTTGCTATG GATTATTGGGGCCAGGGTACCCTTGTGACCGTGAGCTCA 54: PN of VL, GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGA MAB3 GCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTAC encoding CAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGGGCATCCAACCGG SEQ ID NO: 9 GCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTACTACTGCGGCCAG AGCTATAGCTATCCATTTACCTTTGGCCAGGGCACCAAGCTTGAAATTAAA 71: PN of HC, CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG MAB3 AGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGCAGCTATGGTGTGGACTGG encoding GTTCGCCAGGCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGT SEQ ID NO: 69 GGAGGCACATATTATACTGCTTCTCTCATGGGCAGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG GCCGTGTATTACTGCGCCAAACATGCCTATGGCCATGATGGCGGCTTTGCTATG GATTATTGGGGCCAGGGTACCCTTGTGACCGTGAGCTCAGCTAGCACCAAGGGC CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCC GCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGG AACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGC ACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCA GCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG AGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG CACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTG GTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAG TGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAG GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCC AGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTG ACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG 72: PN of LC, GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGA MAB3 GCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTAC encoding CAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGGGCATCCAACCGG SEQ ID NO: 70 GCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTACTACTGCGGCCAG AGCTATAGCTATCCATTTACCTTTGGCCAGGGCACCAAGCTTGAAATTAAACGT ACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCC AAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGC GTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACC CTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCAC CAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 10: VH, MAB4 EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWVGVIWGG GGTYYASSLMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHAYGHDGGFAM DYWGQGTLVTVSS 7: VL, MAB4 EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIK 73: Heavy EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWVGVIWGG chain, MAB4 GGTYYASSLMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHAYGHDGGFAM DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 66: Light chain, EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR MAB4 ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 55: PN of VH, GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTCTGGGGGGTCCCTG MAB4 AGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGCAGCTATGGTGTGGACTGG encoding GTTCGCCAGGCTCCAGGAAAGGGTCTGGAGTGGGTGGGAGTTATATGGGGTGGT SEQ ID NO: 10 GGAGGCACATATTATGCTTCTTCTCTCATGGGCAGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG GCCGTGTATTACTGCGCCAAACATGCCTATGGCCATGATGGCGGCTTTGCTATG GATTATTGGGGCCAGGGTACCCTTGTGACCGTGAGCTCA 52: PN of VL, GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCTCCAGGAGAAAGA MAB4 GCCACCCTCTCCTGCAGGGCCAGTGAGAGTGTTAGCAGTAATGTAGCCTGGTAC encoding CAGCAGAGACCTGGCCAGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGG SEQ ID NO: 7 GCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGCGGCCAG AGCTATAGCTATCCATTTACCTTTGGCCAGGGCACCAAGCTTGAAATTAAG 74: PN of HC, GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTCTGGGGGGTCCCTG MAB4 AGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGCAGCTATGGTGTGGACTGG encoding GTTCGCCAGGCTCCAGGAAAGGGTCTGGAGTGGGTGGGAGTTATATGGGGTGGT SEQ ID NO: 73 GGAGGCACATATTATGCTTCTTCTCTCATGGGCAGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG GCCGTGTATTACTGCGCCAAACATGCCTATGGCCATGATGGCGGCTTTGCTATG GATTATTGGGGCCAGGGTACCCTTGTGACCGTGAGCTCAGCTAGCACCAAGGGC CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCC GCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGG AACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGC ACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCA GCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG AGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG CACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTG GTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAG TGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAG GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCC AGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTG ACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG 68: PN of LC, GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCTCCAGGAGAAAGA MAB4 GCCACCCTCTCCTGCAGGGCCAGTGAGAGTGTTAGCAGTAATGTAGCCTGGTAC encoding CAGCAGAGACCTGGCCAGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGG SEQ ID NO: 66 GCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGCGGCCAG AGCTATAGCTATCCATTTACCTTTGGCCAGGGCACCAAGCTTGAAATTAAGCGT ACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCC AAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGC GTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACC CTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCAC CAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 12: VH, MAB5 EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWLGVIWGG GGTYYTSSLMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHAYGHDGGFAM DYWGQGTLVTVSS 7: VL, MAB5 EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIK 75: Heavy EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWLGVIWGG chain, MAB5 GGTYYTSSLMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHAYGHDGGFAM DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 66: Light chain, EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR MAB5 ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 56: PN of VH, GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTCTGGGGGGTCCCTG MAB5 AGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGCAGCTATGGTGTGGACTGG encoding GTTCGCCAGGCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGT SEQ ID NO: 12 GGAGGCACATATTATACTTCTTCTCTCATGGGCAGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG GCCGTGTATTACTGCGCCAAACATGCCTATGGCCATGATGGCGGCTTTGCTATG GATTATTGGGGCCAGGGTACCCTTGTGACCGTGAGCTCA 52: PN of VL, GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCTCCAGGAGAAAGA MAB5 GCCACCCTCTCCTGCAGGGCCAGTGAGAGTGTTAGCAGTAATGTAGCCTGGTAC encoding CAGCAGAGACCTGGCCAGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGG SEQ ID NO: 7 GCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGCGGCCAG AGCTATAGCTATCCATTTACCTTTGGCCAGGGCACCAAGCTTGAAATTAAG 76: PN of HC, GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTCTGGGGGGTCCCTG MAB5 AGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGCAGCTATGGTGTGGACTGG encoding GTTCGCCAGGCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGT SEQ ID NO: 75 GGAGGCACATATTATACTTCTTCTCTCATGGGCAGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG GCCGTGTATTACTGCGCCAAACATGCCTATGGCCATGATGGCGGCTTTGCTATG GATTATTGGGGCCAGGGTACCCTTGTGACCGTGAGCTCAGCTAGCACCAAGGGC CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCC GCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGG AACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGC ACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCA GCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG AGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG CACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTG GTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAG TGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAG GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCC AGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTG ACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG 68: PN of LC, GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCTCCAGGAGAAAGA MAB5 GCCACCCTCTCCTGCAGGGCCAGTGAGAGTGTTAGCAGTAATGTAGCCTGGTAC encoding CAGCAGAGACCTGGCCAGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGG SEQ ID NO: 66 GCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGCGGCCAG AGCTATAGCTATCCATTTACCTTTGGCCAGGGCACCAAGCTTGAAATTAAGCGT ACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCC AAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGC GTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACC CTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCAC CAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 14: VH, MAB6 EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWLGVIWGG GGTYYTSSLMARFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHAYGHDGGFAM DYWGQGTLVTVSS 7: VL, MAB6 EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIK 77: Heavy EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWLGVIWGG chain, MAB6 GGTYYTSSLMARFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHAYGHDGGFAM DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 66: Light chain, EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR MAB6 ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 57: PN of VH, GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTCTGGGGGGTCCCTG MAB6 AGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGCAGCTATGGTGTGGACTGG encoding GTTCGCCAGGCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGT SEQ ID NO: 14 GGAGGCACATATTATACTTCTTCTCTCATGGCCAGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG GCCGTGTATTACTGCGCCAAACATGCCTATGGCCATGATGGCGGCTTTGCTATG GATTATTGGGGCCAGGGTACCCTTGTGACCGTGAGCTCA 52: PN of VL, GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCTCCAGGAGAAAGA MAB6 GCCACCCTCTCCTGCAGGGCCAGTGAGAGTGTTAGCAGTAATGTAGCCTGGTAC encoding CAGCAGAGACCTGGCCAGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGG SEQ ID NO: 7 GCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGCGGCCAG AGCTATAGCTATCCATTTACCTTTGGCCAGGGCACCAAGCTTGAAATTAAG 78: PN of HC, GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTCTGGGGGGTCCCTG MAB6 AGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAGCAGCTATGGTGTGGACTGG encoding GTTCGCCAGGCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGT SEQ ID NO: 77 GGAGGCACATATTATACTTCTTCTCTCATGGCCAGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG GCCGTGTATTACTGCGCCAAACATGCCTATGGCCATGATGGCGGCTTTGCTATG GATTATTGGGGCCAGGGTACCCTTGTGACCGTGAGCTCAGCTAGCACCAAGGGC CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCC GCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGG AACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGC ACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCA GCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG AGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG CACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTG GTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAG TGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAG GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCC AGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTG ACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG 68: PN of LC, GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCTCCAGGAGAAAGA MAB6 GCCACCCTCTCCTGCAGGGCCAGTGAGAGTGTTAGCAGTAATGTAGCCTGGTAC encoding CAGCAGAGACCTGGCCAGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGG SEQ ID NO: 66 GCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTACTACTGCGGCCAG AGCTATAGCTATCCATTTACCTTTGGCCAGGGCACCAAGCTTGAAATTAAGCGT ACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCC AAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGC GTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACC CTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCAC CAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 99: VH, MAB7 EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWVGVIWGG GGTYYASSLMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHAYGHDGGFAM DYWGQGTLVTVSS 7: VL, MAB7 EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIK 100: Heavy EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWVGVIWGG chain, MAB7 GGTYYASSLMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHAYGHDGGFAM DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 66: Light chain, EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR MAB7 ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 101: PN of VH, GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGTCCGGCGGCTCTCTG MAB7 AGACTGTCTTGCGCTGCCTCCGGCTTCTCCCTGTCCTCTTACGGCGTGGACTGG encoding GTGCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGGGAGTGATCTGGGGCGGA SEQ ID NO: 99 GGCGGCACCTACTACGCCTCTTCCCTGATGGGCCGGTTCACCATCTCCCGGGAC AACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGACACGCCTACGGCCACGACGGCGGCTTCGCCATG GATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC 102: PN of VL, GAGATCGTGATGACCCAGTCCCCCGCCACCCTGTCTGTGTCTCCCGGCGAGAGA MAB7 GCCACCCTGAGCTGCAGAGCCTCCGAGTCCGTGTCCTCCAACGTGGCCTGGTAT encoding CAGCAGAGACCTGGTCAGGCCCCTCGGCTGCTGATCTACGGCGCCTCTAACCGG SEQ ID NO: 7 GCCACCGGCATCCCTGCCAGATTCTCCGGCTCCGGCAGCGGCACCGACTTCACC CTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCGGCCAG TCCTACTCATACCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAG 103: PN of HC, GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGTCCGGCGGCTCTCTG MAB7 AGACTGTCTTGCGCTGCCTCCGGCTTCTCCCTGTCCTCTTACGGCGTGGACTGG encoding GTGCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGGGAGTGATCTGGGGCGGA SEQ ID NO: 100 GGCGGCACCTACTACGCCTCTTCCCTGATGGGCCGGTTCACCATCTCCCGGGAC AACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGACACGCCTACGGCCACGACGGCGGCTTCGCCATG GATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCTAGCACCAAGGGC CCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCT GCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGG AACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGA ACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCA GCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG TCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG CACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTG GTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAG TGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAG GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCC AGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTG ACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGC AAG 104: PN of LC, GAGATCGTGATGACCCAGTCCCCCGCCACCCTGTCTGTGTCTCCCGGCGAGAGA MAB7 GCCACCCTGAGCTGCAGAGCCTCCGAGTCCGTGTCCTCCAACGTGGCCTGGTAT encoding CAGCAGAGACCTGGTCAGGCCCCTCGGCTGCTGATCTACGGCGCCTCTAACCGG SEQ ID NO: 66 GCCACCGGCATCCCTGCCAGATTCTCCGGCTCCGGCAGCGGCACCGACTTCACC CTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCGGCCAG TCCTACTCATACCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGT ACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCC AAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGC GTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACC CTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCAC CAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 105: VH, EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWVGVIWGG MAB8 GGTYYASSLMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNAYGHDGGFAM DYWGQGTLVTVSS 7: VL, MAB8 EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIK 106: Heavy EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEWVGVIWGG chain, MAB8 GGTYYASSLMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNAYGHDGGFAM DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 66: Light chain, EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQAPRLLIYGASNR MAB8 ATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 107: PN of VH, GAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTGCAGTCAGGCGGTAGCCTG MAB8 AGACTGAGCTGCGCCGCCTCCGGCTTTAGCCTGTCTAGCTACGGCGTGGACTGG encoding GTCCGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTCGGAGTGATCTGGGGCGGA SEQ ID NO: 105 GGCGGAACCTACTACGCCTCTAGCCTGATGGGCCGGTTCACTATCTCTAGGGAC AACTCTAAGAACACCCTGTACCTGCAGATGAACTCACTGAGAGCCGAGGACACC GCCGTCTACTACTGCGCTAGAAACGCCTACGGTCACGACGGCGGCTTCGCTATG GACTACTGGGGTCAGGGCACCCTGGTCACCGTGAGTTCA 102: PN of VL, GAGATCGTGATGACCCAGTCCCCCGCCACCCTGTCTGTGTCTCCCGGCGAGAGA MAB8 GCCACCCTGAGCTGCAGAGCCTCCGAGTCCGTGTCCTCCAACGTGGCCTGGTAT encoding CAGCAGAGACCTGGTCAGGCCCCTCGGCTGCTGATCTACGGCGCCTCTAACCGG SEQ ID NO: 7 GCCACCGGCATCCCTGCCAGATTCTCCGGCTCCGGCAGCGGCACCGACTTCACC CTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCGGCCAG TCCTACTCATACCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAG 108: PN of HC, GAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTGCAGTCAGGCGGTAGCCTG MAB8 AGACTGAGCTGCGCCGCCTCCGGCTTTAGCCTGTCTAGCTACGGCGTGGACTGG encoding GTCCGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTCGGAGTGATCTGGGGCGGA SEQ ID NO: 106 GGCGGAACCTACTACGCCTCTAGCCTGATGGGCCGGTTCACTATCTCTAGGGAC AACTCTAAGAACACCCTGTACCTGCAGATGAACTCACTGAGAGCCGAGGACACC GCCGTCTACTACTGCGCTAGAAACGCCTACGGTCACGACGGCGGCTTCGCTATG GACTACTGGGGTCAGGGCACCCTGGTCACCGTGAGTTCAGCTAGCACTAAGGGC CCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCT GCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGG AACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGA ACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCA GCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG TCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG CACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTG GTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAG TGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAG GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCC AGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTG ACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGC AAG 104: PN of LC, GAGATCGTGATGACCCAGTCCCCCGCCACCCTGTCTGTGTCTCCCGGCGAGAGA MAB8 GCCACCCTGAGCTGCAGAGCCTCCGAGTCCGTGTCCTCCAACGTGGCCTGGTAT encoding CAGCAGAGACCTGGTCAGGCCCCTCGGCTGCTGATCTACGGCGCCTCTAACCGG SEQ ID NO: 66 GCCACCGGCATCCCTGCCAGATTCTCCGGCTCCGGCAGCGGCACCGACTTCACC CTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCGGCCAG TCCTACTCATACCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGT ACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCC AAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGC GTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACC CTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCAC CAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC

The CDRs of the antibodies listed in Table 1 can be determined by well known numbering systems known in the art, including those described herein. Table 2 listed the CDRs that are defined by (1) using the numbering system described in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), NIH publication No. 91-3242; and (2) Chothia, see Al-Lazikani et al., (1997) “Standard conformations for the canonical structures of immunoglobulins,” J. Mol. Biol. 273:927-948.

TABLE 2 Kabat and Chothia CDR Comparison SEQ ID NO: Kabat SEQ ID NO: Chothia CDR CDR (Kabat et al., 1991) CDR (Al-Laikani et al., 1997) MAB1 CDRH1 22: SYGVD 79: GFSLRSY MAB1 CDRH2 62: VIWGGGGTNYNSALMA 80: WGGGG MAB1 CDRH3 29: HAYGHDGGFAMDY 29: HAYGHDGGFAMDY MAB1 CDRL1 63: KASENVDTFVS 81: SENVDTF MAB1 CDRL2 64: GASNRYT 82: GAS MAB1 CDRL3 34: GQSYSYPFT 83: SYSYPF MAB2 CDRH1 22: SYGVD 84: GFSLSSY MAB2 CDRH2 23: VIWGGGGTYYASSVMA 80: WGGGG MAB2 CDRH3 29: HAYGHDGGFAMDY 29: HAYGHDGGFAMDY MAB2 CDRL1 30: RASESVSSNVA 85: SESVSSN MAB2 CDRL2 33: GASNRAT 82: GAS MAB2 CDRL3 34: GQSYSYPFT 83: SYSYPF MAB3 CDRH1 22: SYGVD 84: GFSLSSY MAB3 CDRH2 24: VIWGGGGTYYTASLMG 80: WGGGG MAB3 CDRH3 29: HAYGHDGGFAMDY 29: HAYGHDGGFAMDY MAB3 CDRL1 31: RASQSVSSNLA 86: SQSVSSN MAB3 CDRL2 33: GASNRAT 82: GAS MAB3 CDRL3 34: GQSYSYPFT 83: SYSYPF MAB4 CDRH1 22: SYGVD 84: GFSLSSY MAB4 CDRH2 25: VIWGGGGTYYASSLMG 80: WGGGG MAB4 CDRH3 29: HAYGHDGGFAMDY 29: HAYGHDGGFAMDY MAB4 CDRL1 30: RASESVSSNVA 85: SESVSSN MAB4 CDRL2 33: GASNRAT 82: GAS MAB4 CDRL3 34: GQSYSYPFT 83: SYSYPF MAB5 CDRH1 22: SYGVD 84: GFSLSSY MAB5 CDRH2 26: VIWGGGGTYYTSSLMG 80: WGGGG MAB5 CDRH3 29: HAYGHDGGFAMDY 29: HAYGHDGGFAMDY MAB5 CDRL1 30: RASESVSSNVA 85: SESVSSN MAB5 CDRL2 33: GASNRAT 82: GAS MAB5 CDRL3 34: GQSYSYPFT 83: SYSYPF MAB6 CDRH1 22: SYGVD 84: GFSLSSY MAB6 CDRH2 27: VIWGGGGTYYTSSLMA 80: WGGGG MAB6 CDRH3 29: HAYGHDGGFAMDY 29: HAYGHDGGFAMDY MAB6 CDRL1 30: RASESVSSNVA 85: SESVSSN MAB6 CDRL2 33: GASNRAT 82: GAS MAB6 CDRL3 34: GQSYSYPFT 83: SYSYPF MAB7 CDRH1 22: SYGVD 84: GFSLSSY MAB7 CDRH2 25: VIWGGGGTYYASSLMG 80: WGGGG MAB7 CDRH3 29: HAYGHDGGFAMDY 29: HAYGHDGGFAMDY MAB7 CDRL1 30: RASESVSSNVA 85: SESVSSN MAB7 CDRL2 33: GASNRAT 82: GAS MAB7 CDRL3 34: GQSYSYPFT 83: SYSYPF MAB8 CDRH1 22: SYGVD 84: GFSLSSY MAB8 CDRH2 25: VIWGGGGTYYASSLMG 80: WGGGG MAB8 CDRH3 109: NAYGHDGGFAMDY 109: NAYGHDGGFAMDY MAB8 CDRL1 30: RASESVSSNVA 85: SESVSSN MAB8 CDRL2 33: GASNRAT 82: GAS MAB8 CDRL3 34: GQSYSYPFT 83: SYSYPF

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention that binds to GITR (e.g., SEQ ID NO:1, cellular processed SEQ ID NO:1), is selected from any one of: i) an antibody, antibody fragment, or antigen binding molecule wherein: the heavy chain CDR1 comprises SEQ ID NO:22, the heavy chain CDR2 comprises SEQ ID NO:23, the heavy chain CDR3 comprises SEQ ID NO:29, the light chain CDR1 comprises SEQ ID NO:30, the light chain CDR2 comprises SEQ ID NO:33, and the light chain CDR3 comprises SEQ ID NO:34; ii) an antibody, antibody fragment, or antigen binding molecule wherein: the heavy chain CDR1 comprises SEQ ID NO:22, the heavy chain CDR2 comprises SEQ ID NO:24, the heavy chain CDR3 comprises SEQ ID NO:29, the light chain CDR1 comprises SEQ ID NO:31, the light chain CDR2 comprises SEQ ID NO:33, and the light chain CDR3 comprises SEQ ID NO:34; iii) an antibody, antibody fragment, or antigen binding molecule wherein: the heavy chain CDR1 comprises SEQ ID NO:22, the heavy chain CDR2 comprises SEQ ID NO:25, the heavy chain CDR3 comprises SEQ ID NO:29, the light chain CDR1 comprises SEQ ID NO:30, the light chain CDR2 comprises SEQ ID NO:33, and the light chain CDR3 comprises SEQ ID NO:34; iv) an antibody, antibody fragment, or antigen binding molecule wherein: the heavy chain CDR1 comprises SEQ ID NO:22, the heavy chain CDR2 comprises SEQ ID NO:26, the heavy chain CDR3 comprises SEQ ID NO:29, the light chain CDR1 comprises SEQ ID NO:30, the light chain CDR2 comprises SEQ ID NO:33, and the light chain CDR3 comprises SEQ ID NO:34; v) an antibody, antibody fragment, or antigen binding molecule wherein: the heavy chain CDR1 comprises SEQ ID NO:22, the heavy chain CDR2 comprises SEQ ID NO:27, the heavy chain CDR3 comprises SEQ ID NO:29, the light chain CDR1 comprises SEQ ID NO:30, the light chain CDR2 comprises SEQ ID NO:33, and the light chain CDR3 comprises SEQ ID NO:34; and vi) an antibody, antibody fragment, or antigen binding molecule wherein: the heavy chain CDR1 comprises SEQ ID NO:22, the heavy chain CDR2 comprises SEQ ID NO:25, the heavy chain CDR3 comprises SEQ ID NO:109, the light chain CDR1 comprises SEQ ID NO:30, the light chain CDR2 comprises SEQ ID NO:33, and the light chain CDR3 comprises SEQ ID NO:34. In some embodiments, the antibodies or antibody fragments are humanized. In particular embodiments the antibodies or antibody fragments comprise a human constant region. In some embodiments the antibodies or antibody fragments comprise an IgG Fc region. In certain embodiments the antibody or antigen binding fragment is glycosylated. In some embodiments the antibodies or antibody fragments are modified or expressed in a modified cell, wherein such modification results in increased FcR effector function of the antibody or antibody fragment. In certain embodiments the antibody or antigen fragment induces an elevated Teff:Treg ratio in vivo. In some embodiments the antibody or antibody fragment induces a potentiated immune response in vivo. In some embodiments when the antibody or antibody fragment is cross linked to a second antibody or antibody fragment it is an agonist of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO3.

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:16 and comprise a light chain variable region having at least 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:17.

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:6 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:7.

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:8 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:9.

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:10 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:7.

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:12 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:7.

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:14 and comprise a light chain polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:7.

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:99 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:7.

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:105 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:7.

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:61 and comprise a light chain polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:59.

Over their full length, the anti-GITR antibodies of the present invention generally can have an overall constant region (e.g., IgG1) amino acid sequence identity of at least about 85%, for example, at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to human constant region, e.g., IgG1/kappa constant region amino acid sequences. For example, the heavy chain of the anti-GITR antibodies can have at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the human IgG1 constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:20). In one embodiment, the last amino acid, lysine (K), is substituted with arginine (R). The light chain of the anti-GITR antibodies can have at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the human kappa light chain constant region

(SEQ ID NO: 21) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC. In some embodiments, amino acids within the constant regions are added, deleted, or substituted.

In some embodiments, such antibody is a human or humanized antibody. The VH, VL, full length light chain, and full length heavy chain sequences (amino acid sequences and the nucleotide sequences encoding the amino acid sequences) can be “mixed and matched” to create other GITR-binding antibodies of the invention. Such “mixed and matched” GITR-binding antibodies can be tested using the binding assays known in the art (e.g., ELISAs, and other assays described in the Example section) to confirm activity. When chains are mixed and matched, a VH sequence from a particular VH/VL pairing should be replaced with a structurally similar VH sequence. Likewise a full length heavy chain sequence from a particular full length heavy chain/full length light chain pairing should be replaced with a structurally similar full length heavy chain sequence. Likewise, a VL sequence from a particular VH/VL pairing should be replaced with a structurally similar VL sequence. Likewise a full length light chain sequence from a particular full length heavy chain/full length light chain pairing should be replaced with a structurally similar full length light chain sequence. Accordingly, in one aspect, the invention provides an isolated monoclonal antibody or antibody fragment having: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:6, 8, 10, 12, 14, 99 and 105; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:7 and 9; wherein the antibody specifically binds to GITR.

In some embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain sequence selected from any of SEQ ID NO:65, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:100 and SEQ ID NO:106; and comprise a light chain polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain of SEQ ID NO:66 or SEQ ID NO:70. In certain embodiments, the anti-GITR antibodies or antibody fragments of the invention comprise a heavy chain polypeptide selected from any of SEQ ID NO:65, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:100 and SEQ ID NO:106; and comprise a light chain polypeptide of SEQ ID NO:66 or SEQ ID NO:70.

For identified amino acid sequences less than 20 amino acids in length, one or two conservative amino acid residue substitutions can be tolerated while still retaining the desired specific binding and/or agonist activity.

Anti-GITR antibodies and antibody fragments of the present invention generally will bind GITR, including isoform 1(SEQ ID NO:1), isoform 2(SEQ ID NO:2) and isoform 3(SEQ ID NO:3), with an equilibrium dissociation constant (K_(D)) of less than about 10⁻⁸M or 10⁻⁹ M, for example, or less than about 10⁻¹⁰ M or 10⁻¹¹ M, and in some embodiments, less than about 10⁻¹² M or 10⁻¹³ M.

Antibodies that Bind to the Same Epitope

The present invention provides antibodies and antibody fragments that bind to an epitope comprising the cysteine-rich domain 1 (“CRD1”, SEQ ID NO:4: CGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDC) and the cysteine-rich domain 2 (“CRD2”, SEQ ID NO:5: MCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQC) of human GITR, and wherein the antibody, antibody fragment, or the antigen binding molecule is an agonist of hGITR, and wherein the antibody, antibody fragment, or the antigen binding molecule optionally has an intact or increased FcR effector function. In some embodiments, an antibody, antibody fragment, or the antigen binding molecule binds to an epitope comprising SEQ ID NO:88) of human GITR. In some embodiments an epitope comprises residues within SEQ ID NO:88. In some embodiments an epitope comprises amino acid residues within residues 34-72 and 78 of human GITR, where such antibodies and antibody fragments are agonists of hGITR.

The present invention also provides antibodies and antibody fragments that bind to the same epitope as do the GITR-binding antibodies described in Table 1. Additional antibodies and antibody fragments can therefore be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention in GITR binding assays. The ability of a test antibody to inhibit the binding of antibodies and antibody fragments of the present invention to a GITR protein (e.g., human GITR) demonstrates that the test antibody can compete with that antibody or antibody fragment for binding to hGITR; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on the GITR protein as the antibody or antibody fragment with which it competes. In a certain embodiment, the antibody that binds to the same epitope on hGITR as the antibodies or antibody fragments of the present invention is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

Engineered and Modified Antibodies

An antibody or antibody fragment of the invention further can be prepared using an antibody having one or more of the CDRs and/or VH and/or VL sequences shown herein (e.g., Table 1) as starting material to engineer a modified antibody or antibody fragment, which modified antibody may have altered properties from the starting antibody. An antibody or antibody fragment can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody or antibody fragment can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific antibody by constructing expression vectors that include CDR sequences from the specific antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. et al., 1989 Proc. Natl. Acad., U.S.A. 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

Accordingly, another embodiment of the invention pertains to an isolated monoclonal antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising CDR1 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOS:22, 79, and 84; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOS:23, 24, 25, 26, 27, 62, and 80; CDR3 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOS:29, 34 and 109, respectively; and a light chain variable region having CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOS:30, 31, 63, 81, 85, and 86; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOS:33, 64, and 82; and CDR3 sequences consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS:34 and 83; respectively. Thus, such antibodies contain the VH and VL CDR sequences of monoclonal antibodies, yet may contain different framework sequences from these antibodies. In certain embodiments, the isolated antibodies or antibody fragments comprise sequences that have amino acid sequence identity of at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to the corresponding sequences in this paragraph.

Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBase” human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al., 1992 J. fol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. J Immunol. 24:827-836.

An example of framework sequences for use in the antibodies of the invention are those that are structurally similar to the framework sequences used by selected antibodies of the invention, e.g., consensus sequences and/or framework sequences used by monoclonal antibodies of the invention. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3 sequences, can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acid residues within the VH and/or VL CDR1, CDR2, and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, known as “affinity maturation.” Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples and/or alternative or additional assays known in the art. Conservative modifications can be introduced. The mutations may be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

Engineered antibodies or antibody fragments of the invention include those in which modifications have been made to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “backmutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be “backmutated” to the germline sequence by, for example, site-directed mutagenesis. Such “backmutated” antibodies are also intended to be encompassed by the invention.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

When present, the constant regions of the anti-GITR antibodies or antibody fragments can be any type or subtype, as appropriate, and can be selected to be from the species of the subject to be treated by the present methods (e.g., human, non-human primate or other mammal, for example, agricultural mammal (e.g., equine, ovine, bovine, porcine, camelid), domestic mammal (e.g., canine, feline) or rodent (e.g., rat, mouse, hamster, rabbit). In some embodiments the anti-GITR antibodies are engineered to generate humanized or Humaneered® antibodies. In some embodiments, the constant region isotype is IgG, for example, IgG1, IgG2, IgG3, IgG4. In certain embodiments the constant region isotype is IgG₁.

In addition or alternative to modifications made within the framework or CDR regions, antibodies or antibody fragments of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody or antibody fragment of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody or antibody fragment.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody or antibody fragment.

In another embodiment, the Fc hinge region of an antibody is mutated to alter the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to increase the biological half life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor (FcR) or the C1 component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

Antibodies containing such mutations mediate reduced or no antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, amino acid residues L234 and L235 of the IgG1 constant region are substituted to Ala234 and Ala235. In some embodiments, amino acid residue N267 of the IgG1 constant region is substituted to Ala267.

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.

In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fcγ receptor by modifying one or more amino acids. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604).

In still another embodiment, glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for “antigen’. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Grafting Antigen-Binding Domains into Alternative Frameworks or Scaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can be employed so long as the resulting polypeptide includes at least one binding region which specifically binds to GITR. Such frameworks or scaffolds include the 5 main idiotypes of human immunoglobulins, or fragments thereof, and include immunoglobulins of other animal species, preferably having humanized aspects. Single heavy-chain antibodies such as those identified in camelids are of particular interest in this regard. Novel frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.

In one aspect, the invention pertains to generating non-immunoglobulin based antibodies using non-immunoglobulin scaffolds onto which CDRs of the invention can be grafted. Known or future non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for the target GITR protein (e.g., human and/or cynomolgus GITR). Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, Mass.), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, Mass., and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.), maxybodies (Avidia, Inc., Mountain View, Calif.), Protein A (Affibody AG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

The fibronectin scaffolds are based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (see U.S. Pat. No. 6,818,418). These fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprises the entire antigen recognition unit in camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomization and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the invention using standard cloning techniques.

The ankyrin technology is based on using proteins with ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two anti-parallel α-helices and a β-turn. Binding of the variable regions is mostly optimized by using ribosome display.

Avimers are derived from natural A-domain containing protein such as LRP-1. These domains are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on A-domains. Avimers consist of a number of different “A-domain” monomers (2-10) linked via amino acid linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, U.S. Patent Application Publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.

Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibody molecules mimic antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of affibody molecules is similar to that of an antibody.

Anticalins are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, being just marginally bigger than a single immunoglobulin domain. The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains. The binding site can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity. One protein of lipocalin family, the bilin-binding protein (BBP) of Pieris Bras sicae has been used to develop anticalins by mutagenizing the set of four loops. One example of a patent application describing anticalins is in PCT Publication No. WO 199916873.

Affilin molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules. New affilin molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are employed, one of which is gamma crystalline, a human structural eye lens protein and the other is “ubiquitin” superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of gamma crystalline derived proteins are described in WO200104144 and examples of “ubiquitin-like” proteins are described in WO2004106368.

Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-like molecules (MW 1-2 kDa) mimicking beta-hairpin secondary structures of proteins, the major secondary structure involved in protein-protein interactions.

Human or Humanized Antibodies

The present invention provides engineered human antibodies that specifically bind to GITR protein (e.g., human GITR). Compared to the chimeric, primatized, or humanized antibodies, the human GITR-binding antibodies of the invention have further reduced antigenicity when administered to human subjects.

The human GITR-binding antibodies can be generated using methods that are known in the art. For example, the Humaneered® technology platform (KaloBios, Sout San Francisco, Calif.) was used to convert non-human antibodies into engineered human antibodies. U.S. Patent Publication No. 20050008625 describes an in vivo method for replacing a nonhuman antibody variable region with a human variable region in an antibody while maintaining the same or providing better binding characteristics relative to that of the nonhuman antibody. The method relies on epitope guided replacement of variable regions of a non-human reference antibody with a fully human antibody. The resulting human antibody is generally unrelated structurally to the reference nonhuman antibody, but binds to the same epitope on the same antigen as the reference antibody.

The anti-GITR antibodies of the invention are based on engineered human antibodies with V-region sequences having substantial amino acid sequence identity to human germline V region sequences while retaining the specificity and affinity of a reference antibody. See, U.S. Patent Publication No. 2005/0255552 and U.S. Patent Publication No. 2006/0134098, both of which are hereby incorporated herein by reference. The process of improvement identifies minimal sequence information required to determine antigen-binding specificity from the variable region of a reference antibody, and transfers that information to a library of human partial V-region gene sequences to generate an epitope-focused library of human antibody V regions. A microbial-based secretion system can be used to express members of the library as antibody Fab fragments and the library is screened for antigen-binding Fabs, for example, using a colony-lift binding assay. See, e.g., U.S. Patent Publication No. 2007/0020685. Positive clones can be further characterized to identify those with the highest affinity. The resultant engineered human Fabs retain the binding specificity of the parent, reference anti-GITR antibody, typically have equivalent or higher affinity for antigen in comparison to the parent antibody, and have V-regions with a high degree of sequence identity compared with human germ-line antibody V-regions.

The minimum binding specificity determinant (BSD) required to generate the epitope-focused library is typically represented by a sequence within the heavy chain CDR3 (“CDRH3”) and a sequence within the light chain of CDR3 (“CDRL3”). The BSD can comprise a portion or the entire length of a CDR3. The BSD can be comprised of contiguous or non-contiguous amino acid residues. In some cases, the epitope-focused library is constructed from human V-segment sequences linked to the unique CDR3-FR4 region from the reference antibody containing the BSD and human germ-line J segment sequences (see, U.S. Patent Publication No. 2005/0255552). Alternatively, the human V segment libraries can be generated by sequential cassette replacement in which only part of the reference antibody V segment is initially replaced by a library of human sequences. The identified human “cassettes” supporting binding in the context of residual reference antibody amino acid sequences are then recombined in a second library screen to generate completely human V segments (see, U.S. Patent Publication No. 2006/0134098).

In each case, paired heavy and light chain CDR3 segments, CDR3-FR4 segments, or J segments, containing specificity determinants from the reference antibody, are used to constrain the binding specificity so that antigen-binders obtained from the library retain the epitope-specificity of the reference antibody. Additional maturational changes can be introduced in the CDR3 regions of each chain during the library construction in order to identify antibodies with optimal binding kinetics. The resulting engineered human antibodies have V-segment sequences derived from the human germ-line libraries, retain the short BSD sequence from within the CDR3 regions and have human germ-line framework 4 (FR4) regions.

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) family including new world members such as llama species (e.g., Lama paccos, Lama glama, and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects. Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a “camelid nanobody”. See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be “humanized”. Thus the natural low antigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus yet another consequence of small size is that a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.

The low molecular weight and compact size further result in camelid nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that camelid nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitated drug transport across the blood brain barrier. See U.S. patent application 20040161738 published Aug. 19, 2004. These features combined with the low antigenicity to humans indicate great therapeutic potential. Further, these molecules can be expressed in prokaryotic cells such as E. coli and are expressed as fusion proteins with bacteriophage and are functional.

Accordingly, a feature of the present invention is a camelid antibody or nanobody having high affinity for GITR. In certain embodiments herein, the camelid antibody or nanobody is naturally produced in the camelid animal, i.e., is produced by the camelid following immunization with GITR or a peptide fragment thereof, using techniques described herein for other antibodies. Alternatively, the GITR-binding camelid nanobody is engineered, i.e., produced by selection for example from a library of phage displaying appropriately mutagenized camelid nanobody proteins using panning procedures with GITR as a target as described in the examples herein. Engineered nanobodies can further be customized by genetic engineering to have a half life in a recipient subject of from 45 minutes to two weeks. In a specific embodiment, the camelid antibody or nanobody is obtained by grafting the CDRs sequences of the heavy or light chain of the human antibodies of the invention into nanobody or single domain antibody framework sequences, as described for example in PCT/EP93/02214. In some embodiments, the present invention provides multivalent camelid antibody or nanobody, according the methods described below.

Multivalent Antibodies

In another aspect, provided are multivalent molecules (monospecific, bispecific, or multispecific) comprising a GITR-binding antibody, or a fragment thereof, of the invention. In an embodiment an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domains sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).

An antibody of the invention, or antigen-binding regions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a multivalent molecule that binds to at least two different binding sites (which may be the same or different target sites or molecules). As used herein, a “derivatized” antibody molecule is one that has been modified. Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinity ligand such as biotin. Accordingly, the antibody molecules of the invention are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag). In other embodiments, an antibody of the invention (e.g., a monospecific, bispecific, or multispecific antibody molecule) is covalently linked, e.g., fused, to another partner e.g., a protein e.g., one, two or more cytokines, e.g., as a fusion molecule for example a fusion protein. In other embodiments, the fusion molecule comprises one or more proteins, e.g., one, two or more cytokines. In some embodiments the antibody of the invention is derivatized or functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to more than one other functional molecule to generate multivalent molecules that bind to two or more different binding sites which are the same or different binding sites on the same target molecule. In certain embodiments, the multivalent binding sites are the same. In some embodiments the antibody of the invention is derivatized or linked to more than one other functional molecule to generate multi-specific molecules that bind two or more different binding sites on at least two target molecules; such multi-specific molecules are also intended to be encompassed by the term “bispecific molecule” or “multispecific” as used herein. To create a bispecific molecule of the invention, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a multivalent molecule results. The present invention includes bispecific molecules comprising at least one first binding specificity for GITR and a second binding specificity for a second target epitope. For example, the second target epitope is another epitope of GITR different from the first target epitope. Additionally, for the invention in which the molecule is multi-specific, in some embodiments the molecule further includes a third binding specificity, in addition to the first and second target epitope. In an embodiment a multispecific antibody molecule comprises a third, fourth or fifth immunoglobulin variable domain. In an embodiment, a multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or tetraspecific antibody molecule.

In one embodiment, the bispecific molecules of the invention comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab′, F(ab′)2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Pat. No. 4,946,778.

Diabodies are bivalent, bispecific molecules in which VH and VL domains are expressed on a single polypeptide chain, connected by a linker that is too short to allow for pairing between the two domains on the same chain. The VH and VL domains pair with complementary domains of another chain, thereby creating two antigen binding sites (see e.g., Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994 Structure 2:1121-1123). Diabodies can be produced by expressing two polypeptide chains with either the structure VHA-VLB and VHB-VLA (VH-VL configuration), or VLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. Most of them can be expressed in soluble form in bacteria. Single chain diabodies (scDb) are produced by connecting the two diabody-forming polypeptide chains with linker of approximately 15 amino acid residues (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(3-4):128-30; Wu et al., 1996 Immunotechnology, 2(1):21-36). scDb can be expressed in bacteria in soluble, active monomeric form (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(34): 128-30; Wu et al., 1996 Immunotechnology, 2(1):21-36; Pluckthun and Pack, 1997 Immunotechnology, 3(2): 83-105; Ridgway et al., 1996 Protein Eng., 9(7):617-21). A diabody can be fused to Fc to generate a “di-diabody” (see Lu et al., 2004 J. Biol. Chem., 279(4):2856-65).

Other antibodies which can be employed in the bispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.

The bispecific and/or multivalent molecules of the present invention can be prepared by conjugating the constituent binding specificities, using methods known in the art. For example, each binding specificity of the bispecific and/or multivalent molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al., 1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When binding specificities are antibodies, they can be conjugated by sulfhydryl bonding of the constant domain hinge regions of the two heavy chains. In a particular embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, for example one, prior to conjugation.

Alternatively, binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and/or multivalent molecule is a mAb×mAb, mAb×Fab, Fab×F(ab′)2 or ligand x Fab fusion protein. A bispecific and/or multivalent molecule of the invention can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may comprise at least two single chain molecules. Methods for preparing bispecific molecules are described for example in U.S. Pat. No. 5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No. 5,482,858.

Binding of bispecific and/or multivalent molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

Antibodies with Extended Half Life

The present invention provides for antibodies and antibody fragments that specifically bind to GITR protein which have an extended half-life in vivo.

Many factors may affect a protein's half life in vivo. For examples, kidney filtration, metabolism in the liver, degradation by proteolytic enzymes (proteases), and immunogenic responses (e.g., protein neutralization by antibodies and uptake by macrophages and dentritic cells). A variety of strategies can be used to extend the half life of the antibodies of the present invention. For example, by chemical linkage to polyethyleneglycol (PEG), reCODE PEG, antibody scaffold, polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, and carbohydrate shields; by genetic fusion to proteins binding to serum proteins, such as albumin, IgG, FcRn, and transferring; by coupling (genetically or chemically) to other binding moieties that bind to serum proteins, such as nanobodies, Fabs, DARPins, avimers, affibodies, and anticalins; by genetic fusion to rPEG, albumin, domain of albumin, albumin-binding proteins, and Fc; or by incorporation into nancarriers, slow release formulations, or medical devices.

To prolong the serum circulation of antibodies in vivo, inert polymer molecules such as high molecular weight PEG can be attached to the antibodies or a fragment thereof with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of the antibodies or via epsilon-amino groups present on lysine residues. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by size-exclusion or by ion-exchange chromatography. PEG-derivatized antibodies can be tested for binding activity as well as for in vivo efficacy using methods well-known to those of skill in the art, for example, by immunoassays described herein. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Other modified pegylation technologies include reconstituting chemically orthogonal directed engineering technology (ReCODE PEG), which incorporates chemically specified side chains into biosynthetic proteins via a reconstituted system that includes tRNA synthetase and tRNA. This technology enables incorporation of more than 30 new amino acids into biosynthetic proteins in E. coli, yeast, and mammalian cells. The tRNA incorporates a nonnative amino acid any place an amber codon is positioned, converting the amber from a stop codon to one that signals incorporation of the chemically specified amino acid.

Recombinant pegylation technology (rPEG) can also be used for serum halflife extension. This technology involves genetically fusing a 300-600 amino acid unstructured protein tail to an existing pharmaceutical protein. Because the apparent molecular weight of such an unstructured protein chain is about 15-fold larger than its actual molecular weight, the serum halflife of the protein is greatly increased. In contrast to traditional PEGylation, which requires chemical conjugation and repurification, the manufacturing process is greatly simplified and the product is homogeneous.

Polysialytion is another technology, which uses the natural polymer polysialic acid (PSA) to prolong the active life and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). When used for protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment on conjugation. This increases the active life of the therapeutic protein in the circulation and prevents it from being recognized by the immune system. The PSA polymer is naturally found in the human body. It was adopted by certain bacteria which evolved over millions of years to coat their walls with it. These naturally polysialylated bacteria were then able, by virtue of molecular mimicry, to foil the body's defence system. PSA, nature's ultimate stealth technology, can be easily produced from such bacteria in large quantities and with predetermined physical characteristics. Bacterial PSA is completely non-immunogenic, even when coupled to proteins, as it is chemically identical to PSA in the human body.

Another technology include the use of hydroxyethyl starch (“HES”) derivatives linked to antibodies. HES is a modified natural polymer derived from waxy maize starch and can be metabolized by the body's enzymes. HES solutions are usually administered to substitute deficient blood volume and to improve the rheological properties of the blood. Hesylation of an antibody enables the prolongation of the circulation half-life by increasing the stability of the molecule, as well as by reducing renal clearance, resulting in an increased biological activity. By varying different parameters, such as the molecular weight of HES, a wide range of HES antibody conjugates can be customized.

Antibodies having an increased half-life in vivo can also be generated introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into an IgG constant domain, or FcRn binding fragment thereof (preferably a Fc or hinge Fc domain fragment). See, e.g., International Publication No. WO 98/23289; International Publication No. WO 97/34631; and U.S. Pat. No. 6,277,375.

Further, antibodies can be conjugated to albumin in order to make the antibody or antibody fragment more stable in vivo or have a longer half life in vivo. The techniques are well-known in the art, see, e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 413,622.

The strategies for increasing half life is especially useful in nanobodies, fibronectin-based binders, and other antibodies or proteins for which increased in vivo half life is desired.

Antibody Conjugates

The present invention provides antibodies or fragments thereof that specifically bind to a GITR protein recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, preferably to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to generate fusion proteins. In particular, the invention provides fusion proteins comprising an antigen-binding fragment of an antibody described herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain, or a VL CDR) and a heterologous protein, polypeptide, or peptide. Methods for fusing or conjugating proteins, polypeptides, or peptides to an antibody or an antibody fragment are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.

Additional fusion proteins may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of these patents and publications are hereby incorporated by reference in its entirety). Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. A polynucleotide encoding an antibody or fragment thereof that specifically binds to a GITR protein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies or fragments thereof can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine (HHHHHH SEQ ID NO:11) peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine (SEQ ID NO:11) provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin (“HA”) tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the “flag” tag.

An antibody molecules may be conjugated to another molecular entity, typically a diagnostic, detectable, or a therapeutic (e.g., a cytotoxic or cytostatic) agent or moiety. Radioactive isotopes can be used in diagnostic or therapeutic applications. In other embodiments, antibodies of the present invention or fragments thereof conjugated to a diagnostic or detectable agent. Such antibodies can be useful for monitoring or prognosing the onset, development, progression and/or severity of a disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Such diagnosis and detection can accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidinlbiotin and avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as, but not limited to, iodine (131I, 125I, 123I, and 121I,), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In,), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Tin; and positron emitting metals using various positron emission tomographies, and noradioactive paramagnetic metal ions. Radioactive isotopes can be used in diagnostic or therapeutic applications.

The present invention further encompasses an antibody or fragment thereof conjugated to a therapeutic moiety or drug moiety that modifies a given biological effect or response and uses of antibodies or fragments thereof conjugated to a therapeutic moiety. Therapeutic moieties or drug moieties are not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein, peptide, or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, an anti-angiogenic agent; or, a biological response modifier such as, for example, a lymphokine. An antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.

For example, an antibody can be conjugated to therapeutic moieties such as a radioactive metal ion, such as alph-emiters such as 213Bi or macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, 131In, 131LU, 131Y, 131Ho, 131Sm, to polypeptides. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule. Such linker molecules include for example, glycine linkers e.g., GGGGS (SEQ ID NO:15), which may optionally be repeated, e.g., GGGGSGGGGSGGGGS (SEQ ID NO:18), or other linkers are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties. Examples of other therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, l-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846, 545) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclinies (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids).

Techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol. Rev. 62:119-58.

Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Polynucleotides Encoding Agonist Anti-GITR Antibodies

Anti-GITR antibodies, antigen binding molecules, and fragments thereof, can be produced by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies can be obtained by, e.g., hybridoma or recombinant production. Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

The invention further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementary determining regions as described herein. In some embodiments, the polynucleotide encoding the heavy chain variable regions comprises a sequence having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:101, and SEQ ID NO:107. In some embodiments, the polynucleotide encoding the light chain variable regions comprises a sequence having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:102.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:67. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:68.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:72. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected of SEQ ID NO:73.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:74. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:68.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:76. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:68.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:78. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:68.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO:103. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:104.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:108. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:104.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:60. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:58.

The polynucleotides of the invention can encode only the variable region sequence of an anti-GITR antibody. They can also encode both a variable region and a constant region of the antibody. Some of the polynucleotide sequences encode a polypeptide that comprises variable regions of both the heavy chain and the light chain of one of the exemplified mouse anti-GITR antibody. Some other polynucleotides encode two polypeptide segments that respectively are substantially identical to the variable regions of the heavy chain and the light chain of one of the mouse antibodies.

The polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described herein) encoding an anti-GITR antibody or its binding fragment. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method of U.S. Pat. No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press, NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

Also provided in the invention are expression vectors and host cells for producing the anti-GITR antibodies described above. Various expression vectors can be employed to express polynucleotides encoding the anti-GITR antibody chains, fragments, or binding fragments. Both viral-based and nonviral expression vectors can be used to produce the antibodies in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat Genet 15:345, 1997). For example, nonviral vectors useful for expression of the anti-GITR polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C (Invitrogen, San Diego, Calif.), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adenoassociated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides encoding an anti-GITR antibody chain or fragment. In some embodiments, an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions. Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under noninducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of an anti-GITR antibody chain or fragment. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.

Expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted anti-GITR antibody sequences. More often, the inserted anti-GITR antibody sequences are linked to a signal sequences before inclusion in the vector. Vectors to be used to receive sequences encoding anti-GITR antibody light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies or fragments thereof. Typically, such constant regions are human.

Host cells for harboring and expressing the anti-GITR antibody chains can be either prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Other microbes, such as yeast, can also be employed to express anti-GITR polypeptides of the invention. Insect cells in combination with baculovirus vectors can also be used.

In some preferred embodiments, mammalian host cells are used to express and produce the anti-GITR polypeptides of the present invention. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes (e.g., the myeloma hybridoma clones as described in the Examples) or a mammalian cell line harboring an exogenous expression vector (e.g., the SP2/0 myeloma cells exemplified below). These include any normal mortal or normal or abnormal immortal animal or human cell. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including the CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see generally Sambrook et al., supra). Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will often be desired. For example, cell lines which stably express anti-GITR antibody chains or binding fragments can be prepared using expression vectors of the invention which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type.

Assays for Identifying Agonist Anti-GITR Antibodies

Assays for identifying agonist anti-GITR antibodies are known in the art and described herein. Agonist anti-GITR antibodies bind to GITR and promote, induce, stimulate intracellular signaling through GITR.

Binding of the anti-GITR antibodies to GITR can be determined using any method known in the art. For example, binding to GITR can be determined using known techniques, including without limitation ELISA, Western blots, surface plasmon resonance (e.g., BIAcore), and flow cytometry.

Intracellular signaling through GITR can be measured using any method known in the art. For example, activation through GITR promotes NFκB and MAPK signaling. Methods for measuring NFκB and MAPK activation are standard in the art (e.g., use of reporter gene assays, nuclear translocation of NFκB proteins, phosphorylation status of MAPK proteins). Activation through GITR is a co-stimulatory signal that promotes proliferation of activated CD4⁺ and CD8⁺ T cells in the presence of activation through the T-cell receptor (e.g., in the presence of primary or target antigen). Methods for measuring proliferation of cells are standard in the art (e.g., ³H-thymidine incorporation assays, CFSE labeling). Signaling through GITR also co-stimulates activated CD4⁺ and CD8⁺ T cells in the presence of activation through the T-cell receptor to produce cytokines. Signaling through GITR also co-stimulates activated NK cells to produce cytokines. The cytokines can be either or both Th1-type cytokines (e.g., interferon-γ, IL-2 and TNF) and Th2-type cytokines (e.g., IL-4, IL-5, IL-10 and IL-13). Methods for measuring cytokine production are well known in the art (e.g., ELISA assays, ELISpot assays). Activation through GITR may also induce apoptosis. Methods for measuring apoptosis of cells are standard in the art (e.g., Annexin V staining). In performing in vitro assays, test cells or culture supernatant from test cells contacted with the agonist anti-GITR antibodies can be compared to control cells or culture supernatants from control cells that have not been contacted with the agonist anti-GITR antibodies.

The GITR agonist functionalities of the present antibodies can also be measured in vivo. Preferred agonist anti-GITR antibodies have the ability to activate and expand CD4⁺ and CD8⁺ T-cells. The in vivo activation and expansion of CD4⁺ and CD8⁺ T-cells can be measured using any method known in the art, e.g., by flow cytometry. Preferred agonist anti-GITR antibodies can be therapeutically useful in inhibiting tumor growth or promoting tumor retraction. Tumor growth, or inhibition thereof, can be measured using any method known in the art (e.g., visual inspection, calipers, weight, imaging techniques, including MRI). Preferred agonist anti-GITR antibodies can be therapeutically useful in preventing, reducing, inhibiting or eliminating the causative factor of an infectious disease, e.g., a bacterial, fungal, viral or parasitic infection. The efficacy of the agonist anti-GITR antibodies in augmenting a T-cell response or reducing the severity of a disease can be determined by administering a therapeutically effective amount of the antibody to a subject and comparing the subject before and after administration of the antibody. Efficacy of the agonist anti-GITR antibodies in augmenting a T-cell response or reducing the severity of a disease also can be determined by administering a therapeutically effective amount of the antibody to a test subject and comparing the test subject to a control subject who has not been administered the antibody.

Compositions Comprising Agonist Anti-GITR Antibodies

The invention provides pharmaceutical compositions comprising the present anti-GITR antibodies or antigen-binding molecules formulated together with a pharmaceutically acceptable carrier. Optionally, pharmaceutical compositions can additionally contain other therapeutic agents that are suitable for treating or preventing a given disorder. Pharmaceutically acceptable carriers enhance or stabilize the composition, or to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g. by injection or infusion). In another aspect, the present invention provides compositions, e.g., pharmaceutically acceptable compositions, which include an antibody molecule described herein, formulated together with a pharmaceutically acceptable carrier. The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

A pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Route and/or mode of administration vary depending upon the desired results. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. A pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, intranasal, inhalational, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, active compound, e.g., antibody or antigen binding fragment or multivalent molecule of the invention (e.g., monospecific, bispecific or multispecific molecule), may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

An antibody or fragment thereof, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

In some embodiments, the composition is sterile and fluid. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. In certain embodiments compositions can be prepared for storage in a lyophilized form using appropriate excipients (e.g., sucrose)

Pharmaceutical compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention. Applicable methods for formulating the antibodies and determining appropriate dosing and scheduling can be found, for example, in Remington: The Science and Practice of Pharmacy, 21^(st) Ed., University of the Sciences in Philadelphia, Eds., Lippincott Williams & Wilkins (2005); and in Martindale: The Complete Drug Reference, Sweetman, 2005, London: Pharmaceutical Press., and in Martindale, Martindale: The Extra Pharmacopoeia, 31st Edition., 1996, Amer Pharmaceutical Assn, and Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, each of which are hereby incorporated herein by reference. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of the anti-GITR antibody is employed in the pharmaceutical compositions of the invention. The anti-GITR antibodies are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the desired response (e.g., a therapeutic response). In determining a therapeutically or prophylactically effective dose, a low dose can be administered and then incrementally increased until a desired response is achieved with minimal or no undesired side effects. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Actual dosage levels of active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.

The antibody molecules can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. For example, the antibody molecules can be administered by intravenous infusion at a rate of more than 20 mg/min, e.g., 20-40 mg/min, and typically greater than or equal to 40 mg/min to reach a dose of about 35 to 440 mg/m², typically about 70 to 310 mg/m², and more typically, about 110 to 130 mg/m². In embodiments, the antibody molecules can be administered by intravenous infusion at a rate of less than 10 mg/min; preferably less than or equal to 5 mg/min to reach a dose of about 1 to 100 mg/m², preferably about 5 to 50 mg/m², about 7 to 25 mg/m² and more preferably, about 10 mg/m². As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody molecule is 0.1-30 mg/kg, more preferably 1-25 mg/kg. Dosages and therapeutic regimens of the anti-GITR antibody molecule can be determined by a skilled artisan. In certain embodiments, the anti-GITR antibody molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 40 mg/kg, e.g., 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, 1 to 10 mg/kg, 5 to 15 mg/kg, 10 to 20 mg/kg, 15 to 25 mg/kg, or about 3 mg/kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks. In one embodiment, the anti-GITR antibody molecule is administered at a dose from about 10 to 20 mg/kg every other week. The antibody molecule can be administered by intravenous infusion at a rate of more than 20 mg/min, e.g., 20-40 mg/min, and typically greater than or equal to 40 mg/min to reach a dose of about 35 to 440 mg/m², typically about 70 to 310 mg/m², and more typically, about 110 to 130 mg/m². In embodiments, the infusion rate of about 110 to 130 mg/m² achieves a level of about 3 mg/kg. In other embodiments, the antibody molecule can be administered by intravenous infusion at a rate of less than 10 mg/min, e.g., less than or equal to 5 mg/min to reach a dose of about 1 to 100 mg/m², e.g., about 5 to 50 mg/m², about 7 to 25 mg/m², or, about 10 mg/m². In some embodiments, the antibody is infused over a period of about 30 min. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

Co-Formulation with Second Agent

In some embodiments, the pharmacological compositions comprise a mixture of the anti-GITR antibody or antigen binding molecule and a second pharmacological agent. Exemplary second agents for inclusion in mixtures with the present anti-GITR agonist antibody or antigen binding molecule include without limitation primary or target antigens, agents that increase the immunogenicity of a tumor cell, agents that inhibit or suppress co-inhibitory signals.

The anti-GITR antibodies or antigen binding molecules of the invention can be co-formulated (i.e., provided as a mixture or prepared in a mixture) with a primary or target antigen. The target antigen, or vaccine, will depend on the disease condition to be treated. For example, the target antigen may be from a tumor cell, a bacterial cell, a fungus, a virus or a parasite. The target antigen can be in the form of a peptide, a polypeptide, a cell or a polynucleotide, as appropriate.

In one embodiment, the target antigen is from a virus, e.g., selected from the group consisting of: hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV I), herpes simplex type II (HSV II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV I), and human immunodeficiency virus type II (HIV II), any picornaviridae, enteroviruses, caliciviridae, any of the Norwalk group of viruses, togaviruses, such as alphaviruses, flaviviruses, coronaviruses, rabies virus, Marburg viruses, ebola viruses, parainfluenza virus, orthomyxoviruses, bunyaviruses, arenaviruses, reoviruses, rotaviruses, orbiviruses, human T cell leukemia virus type I, human T cell leukemia virus type II, simian immunodeficiency virus, lentiviruses, polyomaviruses, parvoviruses, Epstein Barr virus, human herpesvirus 6, cercopithecine herpes virus 1 (B virus), and poxviruses.

In one embodiment, the target antigen is from a bacterium, e.g., selected from the group consisting of: Neisseria spp, Streptococcus spp, S. mutans, Haemophilus spp., Moraxella spp, Bordetella spp, Mycobacterium spp, Legionella spp, Escherichia spp, Vibrio spp, Yersinia spp, Campylobacter spp, Salmonella spp, Listeria spp., Helicobacter spp, Pseudomonas spp, Staphylococcus spp., Enterococcus spp, Clostridium spp., Bacillus spp, Corynebacterium spp., Borrelia spp., Ehrlichia spp, Rickettsia spp, Chlamydia spp., Leptospira spp., Treponema spp.

In some embodiments, the anti-GITR antibodies or antigen binding molecules are co-formulated in a mixture with a tumor-associated antigen (TAA). The TAA can be an isolated polypeptide or peptide, can be part of an intact cell or part of a tumor cell lysate. The TAAs can be a polynucleotide, for example a naked plasmid or a viral vector comprising a polynucleotide encoding one or more TAAs. Examples of known TAAs include without limitation, melanoma associated antigens (MAGE-1, MAGE-3, TRP-2, melanosomal membrane glycoprotein gp100, gp75 and MUC-1 (mucin-1) associated with melanoma); CEA (carcinoembryonic antigen) which can be associated, e.g., with ovarian, melanoma or colon cancers; folate receptor alpha expressed by ovarian carcinoma; free human chorionic gonadotropin beta (hCGβ) subunit expressed by many different tumors, including but not limited to myeloma; HER-2/neu associated with breast cancer; encephalomyelitis antigen HuD associated with small-cell lung cancer; tyrosine hydroxylase associated with neuroblastoma; prostate-specific antigen (PSA) associated with prostate cancer; CA125 associated with ovarian cancer; and the idiotypic determinants of a B cell lymphoma can generate tumor-specific immunity (attributed to idiotype-specific humoral immune response). Moreover, antigens of human T cell leukemia virus type 1 have been shown to induce specific CTL responses and antitumor immunity against the virus-induced human adult T cell leukemia (ATL). See, e.g., Haupt, et al., Experimental Biology and Medicine (2002) 227:227-237; Ohashi, et al., Journal of Virology (2000) 74(20):9610-9616. Other TAAs are known and find use for co-formulation with the anti-GITR antibodies.

In some embodiments, the anti-GITR antibodies or antigen binding molecules are co-formulated with autologous tumor cells from the patient, or allogeneic tumor cells of the same tissue type from another patient. The tumor cells can be in the form of intact cells, tumor cell lysate, apoptotic tumor cells or total tumor mRNA. The tumor cells can be transfected to express a polypeptide that enhances or augments the immunogenity of the tumor cell in the patient, e.g., transfected to express granulocyte colony stimulating factor (GM-CSF). The tumor cells can be from any cancerous tissue, including without limitation, epithelial cancers or carcinomas, as well as sarcomas and lymphomas. In some embodiments, the cancer is melanoma, ovarian cancer, renal cancer, colorectal cancer, prostate, lung cancer including non-small cell lung cancer (NSCLC), breast cancer, glioma, fibrosarcoma, hematologic cancer, or a head and neck squamous cell carcinoma (HNSCC). See, e.g., Pardee, et al, Immunotherapy (2009) 1(2):249-264, and references discussed therein. In some embodiments, the tumor cell is from, e.g., pancreatic cancer, melanomas, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and cancer of hematological tissues.

In some embodiments, the anti-GITR antibodies or antigen binding molecules are co-formulated with a cytotoxic agent. For example, the anti-GITR antibodies or antigen binding molecules are co-formulated with an agonist antibody or antigen binding molecule that binds to and reduces or depletes CD4+CD25+ regulatory T cells (Treg). Exemplary Treg cell-depleting antibodies or antigen binding molecules bind to CD25 or CCR4. See, Expert Opin Ther Patents (2007) 17(5):567-575, and the references discussed therein.

In some embodiments, the anti-GITR antibodies or antigen binding molecules are co-formulated with an inhibitor of a co-inhibitory signal. Exemplary inhibitors include inhibitors of CTLA-4 and inhibitors of the PD-1/PD-L1 (e.g., B7-H1) interaction. In some embodiments, the anti-GITR antibodies are co-formulated with an antibody that binds to and inhibits CTLA-4. In some embodiments, the anti-GITR antibodies are co-formulated with an antibody that binds to and inhibits TIM3. In some embodiments, the anti-GITR antibodies are co-formulated with an antibody that binds to and inhibits LAG3. In some embodiments, the anti-GITR antibodies are co-formulated with an antibody that binds to and inhibits PD-1. In some embodiments, the anti-GITR antibodies are co-formulated with an antibody that binds to and inhibits B7-H1. See, e.g., Expert Opin Ther Patents (2007) 17(5):567-575; and Melero, et al., Clin Cancer Res (2009) 15(5):1507-1509, and the references discussed therein. In certain embodiments, formulations comprising a bispecific molecule including an anti-GITR antibody or antigen binding molecule and inhibitor of a co-inhibitory signal. In some embodiments, formulations comprise a bispecific molecule including an anti-GITR antibody or antigen binding molecule and an inhibitor of CTLA4. In some embodiments, formulations comprise a bispecific molecule including an anti-GITR antibody or antigen binding molecule and an inhibitor of TIM3. In some embodiments, formulations comprise a bispecific molecule including an anti-GITR antibody or antigen binding molecule and an inhibitor of LAG3. In some embodiments, formulations comprise a bispecific molecule including an anti-GITR antibody or antigen binding molecule and an inhibitor of PD-1/PD-L1. In some embodiments, formulations comprise a bispecific molecule including an anti-GITR antibody or antigen binding molecule and an inhibitor B7H1.

The anti-GITR antibodies or antigen binding molecules can also be co-formulated with one or more immunostimulatory agents. For example, in some embodiments, the anti-GITR antibodies are co-formulated with an immunostimulatory cytokine, for example, IL-7, IL-12 or IL-15. Alternatively, the anti-GITR antibodies or antigen binding molecules can be co-formulated with a second immunostimulatory antibody. For example, the anti-GITR antibodies or antigen binding molecules can also be co-formulated with an agonist antibody or antigen binding molecule of another member of the tumor necrosis factor receptor superfamily. Exemplary secondary immunostimulatory targets include without limitation TNFRSF4 tumor necrosis factor receptor superfamily, member 4 (also known as OX40) or tumor necrosis factor receptor superfamily, member 9 (also known as TNFRSF9, 4-1BB or CD137). See, e.g., Expert Opin Ther Patents (2007) 17(5):567-575; Pardee, et al, Immunotherapy (2009) 1(2):249-264; and Melero, et al., Clin Cancer Res (2009) 15(5):1507-1509, and the references discussed therein.

The anti-GITR antibodies or antigen binding molecules can also be co-formulated with a chemotherapeutic agent. The selected agent will depend on the condition to be treated, e.g., a cancer or an infectious disease, such as a bacterial infection, a fungal infection, a viral infection or a parasitic infection. The anti-GITR antibodies or antigen binding molecules can be co-formulated with a chemotherapeutic known by those of skill to treat the disease condition being treated. Chemotherapeutic agents, e.g., for the treatment of cancers and infectious diseases are known in the art, and are described, e.g., in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., Brunton, Lazo and Parker, Eds., McGraw-Hill (2006); 2010 Physicians' Desk Reference (PDR), 64th Edition, Thomson PDR.

In some embodiments, the anti-GITR antibodies or antigen binding molecules can be co-formulated with an antineoplastic agent. Exemplary antineoplastic agents that find use for mixing in compositions with the anti-GITR antibodies include alkylating agents (e.g., nitrogen mustards, ethyleneimines and methylmelamines, methylhydrazine derivative, alkyl sulfonate, nitrosoureas, triazenes and platinum coordination complexes); antimetabolites (e.g., folic acid analogs, pyrimidine analogs, purine analogs; natural products (e.g., vinca alkaloids, taxanes, epipodophyllotoxins, camptothecins, antibiotics, and anthracenedione). In some embodiments, the anti-GITR antibodies or antigen binding molecules are co-formulated with an antimetabolite antineoplastic agent, e.g., a folic acid analog (e.g., methotrexate, pemetrexed, trimetrexate), a pyrimidine analog (e.g., 5-fluorouracil, capecitabine, cytarabine, gemcitabine), a purine analog (e.g., mercaptopurine, pentostatin, cladribine fludarabine), or mixtures thereof. In some embodiments, the anti-GITR antibodies or antigen binding molecules are co-formulated with an alkylating agent antineoplastic agent, e.g., nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethyleneimines (e.g., altretamine) and methylmelamines (e.g., thiotepa), methylhydrazine derivatives (e.g., procarbazine), alkyl sulfonate (e.g., busulfan), nitrosoureas (e.g., carmustine, streptozocin), triazenes (e.g., dacarbazine, temozolomide) and platinum coordination complexes (e.g., cisplatin, carboplatin, oxaliplatin).

In some embodiments, the anti-GITR antibodies or antigen binding molecules can be co-formulated with an antiviral agent. Exemplary antiviral agents include without limitation anti-herpesvirus agents (e.g., acyclovir, cidofovir, famciclovir, foscarnet, thiovir, fomivirsen, ganciclovir, idoxuridine, penciclovir, trifluridine, valacyclovir, valgenciclovir, resiquimod); anti-influenza agents (e.g., amantadine, oseltamivir, rimantadine, zanamivir, peramivir, E-118958); anti-hepatitis agents (e.g., adeforvir dipivoxil, interferon-alpha, lamivudine, entecavir, clevudine, emtricitabine, telbivudine, tenofovir, viramidine, BILN 2061, NM283) and other antiviral agents (e.g., ribavirin, imiquimod, maribavir, sICAM-1, pleconaril). The antiviral agent can be an antiretroviral agent. Exemplary antiretroviral agents include without limitation zidovudine, didanosine, stavudine, zalcitabine, lamivudine, abacavir, tenofavir, emtricitabine, nevirapine, efavirenz, delavirdine, saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir and enfuvirtide.

In some embodiments, the anti-GITR antibodies or antigen binding molecules can be co-formulated with an antibacterial agent. Exemplary antibacterial agents include without limitation sulfonamides (e.g., sulfanilamide, sulfadiazine, sulfamethoxazole, sulfisoxazole, sulfacetamide), trimethoprim, quinolones (e.g., nalidixic acid, cinoxacin, norfloxacin, ciprofloxacin, ofloxacin, sparfloxacin, fleroxacin, perloxacin, levofloxacin, garenoxacin and gemifloxacin), methenamine, nitrofurantoin, penicillins (e.g., penicillin G, penicillin V, methicilin oxacillin, cloxacillin, dicloxacillin, nafcilin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, and piperacillin), cephalosporins (e.g., cefazolin, cephalexin, cefadroxil, cefoxitin, cefaclor, cefprozil, cefuroxime, cefuroxime acetil, loracarbef, cefotetan, ceforanide, cefotaxime, cefpodoxime proxetil, cefibuten, cefdinir, cefditoren pivorxil, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, and cefepine), carbapenems (e.g., imipenem, aztreonam), and aminoglycosides (e.g., neomycin, kanamycin, streptomycin, gentamicin, toramycin, netilmicin, and amikacin).

In some embodiments, the anti-GITR antibodies or antigen binding molecules can be co-formulated with an anti-parasitic agent. Exemplary anti-parasitic agents include without limitation anti-malarial agents (e.g., quinolines including chloroquine, mefloquine, quinine, quinidine, and primaquine; diaminopyrimidines including pyrimethamine, sulfadoxine, tetracyclines, atovaquone, and proguanil); anti-protozoal agents including amphotericin, chloroquine, eflornithine, emetine, fumagillin, 8-hydroxyquinolines, melarsoprol, metronidazole, miltefosine, nifurtimox, nitazoxanide, paromomycin, pentamidine, sodium stibogluconate, and suramin.

In some embodiments, the anti-GITR antibodies or antigen binding molecules can be co-formulated with an anti-fungal agent. Exemplary anti-fungal agents include without limitation polyenes (e.g., natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, and hamycin), imidazoles (e.g., miconazole, ketoconazole, clotrimazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole), triazoles (e.g., fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole), thiazoles (e.g., abafungin), allylamines (e.g., terbinafine, amorolfine, naftifine, butenafine), echinocandins (e.g., anidulafungin, caspofungin, micafungin), benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine or 5-fluorocytosine, griseofulvin, and haloprogin.

Kits

The anti-GITR compositions of the present invention can be provided in a kit. The anti-GITR antibody, antibody fragment, or antigen binding molecule is generally in a vial or a container. As appropriate, the antibody can be in liquid or dried (e.g., lyophilized) form. The kits can comprise an anti-GITR antibody, antibody fragment, or antigen binding molecule of the invention, as described herein, and optionally also contain a second or third agent. In some embodiments, the kits contain anti-GITR antibody, antibody fragment, or antigen binding molecule of the invention and a pharmaceutically acceptable diluent. The anti-GITR antibodies, antibody fragments, or antigen binding molecules can be provided in the kit with the second or third agents in the same or separate formulations (e.g., as mixtures or in separate containers). The kits can contain aliquots of the anti-GITR antibodies, antibody fragments, or antigen binding molecules that provide for one or more doses. If aliquots for multiple administrations are provided, the doses can be uniform or varied. Varied dosing regimens can be escalating or decreasing, as appropriate. The dosages of the anti-GITR antibody, antibody fragment, or antigen binding molecule and the second agent can be independently uniform or varying. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, an antibody to a label or therapeutic agent, or a radioprotective composition; devices or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

In some embodiments, the kits further contain a target antigen. The target antigen, or vaccine, will depend on the disease condition to be treated. For example, the target antigen may be from a tumor cell, a bacterial cell, a fungus, a parasite or a virus. The target antigen can be in the form of a peptide, a polypeptide, a cell, a polynucleotide (e.g., naked plasmid or viral vector) as appropriate. In some embodiments, the target antigen is a tumor associated antigen. Exemplary target antigens are discussed herein; others known in the art also find use.

In some embodiments, the kits further contain a cytotoxic agent. For example, the kits can contain an agonist antibody or antigen binding molecule that binds to and reduces or depletes CD4+CD25+ regulatory T cells (Treg). Exemplary Treg cell-depleting antibodies or antigen binding molecules bind to CD25 or CCR4. See, Expert Opin Ther Patents (2007) 17(5):567-575, and the references discussed therein.

In some embodiments, the kits further contain an inhibitor of a co-inhibitory signal. Exemplary inhibitors include inhibitors of CTLA-4, LAG3, TIM3, and/or inhibitors of the PD-1/PD-L1 (e.g., B7-H1) interaction. In some embodiments, the kits further contain an antibody that binds to and inhibits CTLA-4. In some embodiments, the kits further contain an antibody that binds to and inhibits LAG3. In some embodiments, the kits further contain an antibody that binds to and inhibits TIM3. In some embodiments, the kits further contain an antibody that binds to and inhibits PD-1. In some embodiments, the kits further contain an antibody that binds to and inhibits B7-H1. See, e.g., Expert Opin Ther Patents (2007) 17(5):567-575; and Melero, et al., Clin Cancer Res (2009) 15(5):1507-1509, and the references discussed therein.

In some embodiments, the kits further contain one or more immunostimulatory agents. For example, in some embodiments, the kits contain an immunostimulatory cytokine, for example, IL-7, IL-12 or IL-15. Alternatively, the kits can contain a second immunostimulatory antibody. For example, the kits can contain an agonist antibody or antigen binding molecule of another member of the tumor necrosis factor receptor superfamily. Exemplary secondary immunostimulatory targets include without limitation TNFRSF4 tumor necrosis factor receptor superfamily, member 4 (also known as OX40) or tumor necrosis factor receptor superfamily, member 9 (also known as TNFRSF9, 4-1BB or CD137). See, e.g., Expert Opin Ther Patents (2007) 17(5):567-575; Pardee, et al, Immunotherapy (2009) 1(2):249-264; and Melero, et al., Clin Cancer Res (2009) 15(5):1507-1509, and the references discussed therein.

In some embodiments, the kits further contain a chemotherapeutic agent. The selected agent will depend on the condition to be treated, e.g., a cancer or an infectious disease, such as a bacterial infection, a fungal infection, a viral infection or a parasitic infection. Exemplary chemotherapy agents include any antineoplastic, antiviral, antibacterial, antiparasitic, and antifungal agents known in the art and described herein.

Methods of Enhancing T Cell Responses Conditions Subject to Treatment or Prevention

The anti-GITR agonist antibodies and antibody fragments of the invention find use in augmenting CD4⁺ T helper and CD8⁺ cytolytic T cell responses in a patient in need thereof. Therefore, the antibodies find use in enhancing or augmenting a T cell response in a patient, e.g., to effect the reduction, reversal, inhibition or prevention of a disease that can be counteracted with an enhanced or augmented immune response. In one aspect, the invention provides methods of enhancing a T cell response in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an anti-GITR agonist antibody or antibody fragment of the invention, as described herein. The invention also provides in one aspect an anti-GITR agonist antibody or antibody fragment for use in enhancing a T cell response in an individual. In a further aspect, the invention provides a composition comprising such an antibody or antibody fragment for use in enhancing a T cell response in an individual.

Conditions subject to treatment include cancers and infectious disease. For therapeutic purposes, the patient may have a cancer or tumor or an infectious disease, e.g., a bacterial, viral, fungal or parasitic infection. For preventative purposes, the patient may be in remission from a cancer or may anticipate being exposed to a bacterial, viral, fungal or parasitic infection. The antibodies can also serve as an adjuvant to enhance or promote or boost an immune response against a primary antigen or a target antigen, e.g., a vaccine.

In some embodiments, the patient has a cancer, is suspected of having a cancer, or is in remission from a cancer. Cancers subject to treatment with the anti-GITR antibodies usually express a tumor-associated antigen (TAA), as described herein. Cancers subject to treatment include without limitation epithelial cancers or carcinomas, as well as sarcomas and lymphomas. In some embodiments, the cancer is melanoma, ovarian cancer, renal cancer, colorectal cancer, prostate, lung cancer including non-small cell lung cancer (NSCLC), breast cancer, glioma, or fibrosarcoma. See, e.g., Pardee, et al, Immunotherapy (2009) 1(2):249-264, and references discussed therein. In some embodiments, the type of cancer is selected from the group consisting of: pancreatic cancer, melanomas, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues and head and neck squamous cell carcinoma (HNSCC).

In one aspect, the invention provides methods of treating tumor growth of a cancer that expresses a tumor associated antigen in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an anti-GITR agonist antibody or antibody fragment of the invention, as described herein. The invention also provides an anti-GITR agonist antibody or antibody fragment of the invention for use in treating tumor growth of a cancer that expresses a tumor associated antigen in an individual. The invention further provides a composition comprising an antibody or antibody fragment of the invention for use in reducing, inhibiting or preventing tumor growth of a cancer that expresses a tumor associated antigen in an individual.

In some embodiments, methods for facilitating the diagnosis or prognosis of cancer in an individual, comprising using an anti-GITR agonist antibody or antibody fragment of the invention for the detection of expression of GITR in or around a tumor in the individual.

In some embodiments, the patient has an infectious disease, for example, a bacterial, viral, fungal or parasitic infection. The anti-GITR agonist antibodies find use in reducing, inhibiting and/or preventing parasites in, e.g., filariasis and leishmaniasis.

In some embodiments, anti-GITR agonist antibodies find use in treatment of viral infections, including without limitation hepatitis virus infection, for example, chronic hepatitis C (HCV) infection, herpes simplex virus (HSV) infection or human immunodeficiency virus (HIV) infection. In some embodiments, anti-GITR agonist antibodies find use in treating a viral infection selected from the group consisting of: hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV I), herpes simplex type II (HSV II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV I), and human immunodeficiency virus type II (HIV II), any picornaviridae, enteroviruses, caliciviridae, any of the Norwalk group of viruses, togaviruses, such as alphaviruses, flaviviruses, coronaviruses, rabies virus, Marburg viruses, ebola viruses, parainfluenza virus, orthomyxoviruses, bunyaviruses, arenaviruses, reoviruses, rotaviruses, orbiviruses, human T cell leukemia virus type I, human T cell leukemia virus type II, simian immunodeficiency virus, lentiviruses, polyomaviruses, parvoviruses, Epstein Barr virus, human herpesvirus 6, cercopithecine herpes virus 1 (B virus), and poxviruses.

In some embodiments, anti-GITR agonist antibodies find use in treating bacterial infections, including without limitation an infection of Neisseria spp, Streptococcus spp, S. mutans, Haemophilus spp., Moraxella spp, Bordetella spp, Mycobacterium spp, Legionella spp, Escherichia spp, Vibrio spp, Yersinia spp, Campylobacter spp, Salmonella spp, Listeria spp., Helicobacter spp, Pseudomonas spp, Staphylococcus spp., Enterococcus spp, Clostridium spp., Bacillus spp, Corynebacterium spp., Borrelia spp., Ehrlichia spp, Rickettsia spp, Chlamydia spp., Leptospira spp., Treponema spp.

Administration of Anti-GITR Antibodies

A physician or veterinarian can start doses of the antibodies or antibody fragments of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, effective doses of the compositions of the present invention vary depending upon many different factors, including the specific disease or condition to be treated, means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy. For administration with an antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. Dosing can be daily, weekly, bi-weekly, monthly, or more or less often, as needed or desired. An exemplary treatment regime entails administration once weekly, once per every two weeks or once a month or once every 3 to 6 months.

In some embodiments, an polynucleotide encoding an anti-GITR antibody, antibody fragment, or antigen binding molecule of the invention is administered. In embodiments where the agent is a nucleic acid, typical dosages can range from about 0.1 mg/kg body weight up to and including about 100 mg/kg body weight, e.g., between about 1 mg/kg body weight to about 50 mg/kg body weight. In some embodiments, about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mg/kg body weight.

The antibody or antibody fragment can be administered in single or divided doses. Antibody or antibody fragment is usually administered on multiple occasions. Intervals between single dosages can be weekly, bi-weekly, monthly or yearly, as needed or desired. Intervals can also be irregular as indicated by measuring blood levels of anti-GITR antibody or antibody fragment in the patient. In some methods, dosage is adjusted to achieve a plasma antibody or antibody fragment concentration of 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, antibody or antibody fragment can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody or antibody fragment in the patient. In general, humanized antibodies show longer half life than that of chimeric antibodies and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

Co-Administration with Additional Agent(s)

In some embodiments, the anti-GITR antibody, antibody fragment, or antigen binding molecule is co-administered with a second or third pharmacological agent. The anti-GITR antibody, antibody fragment, or antigen binding molecule and the second or agent can be administered as a mixture or in separate formulations. The anti-GITR antibody, antibody fragment, or antigen binding molecule and the second or agent can be administered concurrently or sequentially. The anti-GITR antibody, antibody fragment, or antigen binding molecule and the second or agent can be administered via the same route of administration or via different routes of administration, as appropriate. Exemplary second agents and third agents for co-administration with the present anti-GITR agonist antibodies, antibody fragments, or antigen binding molecules include without limitation, primary or target antigens, agents that increase the immunogenicity of a tumor cell, agents that inhibit or suppress co-inhibitory signals. The anti-GITR agonist antibodies, antibody fragments, or antigen binding molecules can also be co-administered with chemotherapeutic used to treat the disease condition being treated, e.g., to enhance the efficacy of the chemotherapeutic agent or to further enhance an immune response against a target antigen. The anti-GITR agonist antibodies, antibody fragments, or antigen binding molecules also find use in combination therapies with established procedures for treating the designated disease condition, e.g., radiation or surgery.

By “a combination” or “in combination with,” it is not intended to imply that the therapy or the therapeutic agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope described herein. The therapeutic agents in the combination can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents. The therapeutic agents or therapeutic protocol can be administered in any order. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutic agent utilized in this combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that additional therapeutic agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

In embodiments, the additional therapeutic agent is administered at a therapeutic or lower-than therapeutic dose. In certain embodiments, the concentration of the second therapeutic agent that is required to achieve inhibition, e.g., growth inhibition, is lower when the second therapeutic agent is administered in combination with the first therapeutic agent, e.g., the anti-GITR antibody molecule, than when the second therapeutic agent is administered individually. In certain embodiments, the concentration of the first therapeutic agent that is required to achieve inhibition, e.g., growth inhibition, is lower when the first therapeutic agent is administered in combination with the second therapeutic agent than when the first therapeutic agent is administered individually. In certain embodiments, in a combination therapy, the concentration of the second therapeutic agent that is required to achieve inhibition, e.g., growth inhibition, is lower than the therapeutic dose of the second therapeutic agent as a monotherapy, e.g., 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90% lower. In certain embodiments, in a combination therapy, the concentration of the first therapeutic agent that is required to achieve inhibition, e.g., growth inhibition, is lower than the therapeutic dose of the first therapeutic agent as a monotherapy, e.g., 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90% lower.

The term “inhibition,” “inhibitor,” or “antagonist” includes a reduction in a certain parameter, e.g., an activity, of a given molecule, e.g., an immune checkpoint inhibitor. For example, inhibition of an activity, e.g., a coinhibitory activity, of at least 5%, 10%, 20%, 30%, 40% or more is included by this term. Thus, inhibition need not be 100%.

The term “activation,” “activator,” or “agonist” includes an increase in a certain parameter, e.g., an activity, of a given molecule, e.g., a costimulatory molecule. For example, increase of an activity, e.g., a costimulatory activity, of at least 5%, 10%, 25%, 50%, 75% or more is included by this term.

The anti-GITR antibodies, antibody fragments, or antigen binding molecules of the invention can be co-administered with a primary or target antigen. The target antigen, or vaccine, will depend on the disease condition to be treated. For example, the target antigen may be from a tumor cell, a bacterial cell, a fungus, a virus or a parasite. The target antigen can be in the form of a peptide, a polypeptide, a cell or a polynucleotide, as appropriate.

In some embodiments, the anti-GITR antibodies, antibody fragments, or antigen binding molecules are co-administered with a target antigen from a virus, e.g., selected from the group consisting of: hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV I), herpes simplex type II (HSV II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV I), and human immunodeficiency virus type II (HIV II), any picornaviridae, enteroviruses, caliciviridae, any of the Norwalk group of viruses, togaviruses, such as alphaviruses, flaviviruses, coronaviruses, rabies virus, Marburg viruses, ebola viruses, parainfluenza virus, orthomyxoviruses, bunyaviruses, arenaviruses, reoviruses, rotaviruses, orbiviruses, human T cell leukemia virus type I, human T cell leukemia virus type II, simian immunodeficiency virus, lentiviruses, polyomaviruses, parvoviruses, Epstein Ban virus, human herpesvirus 6, cercopithecine herpes virus 1 (B virus), and poxviruses.

In some embodiments, the anti-GITR antibodies, antibody fragments, or antigen binding molecules are co-administered with target antigen from a bacterium, e.g., selected from the group consisting of: Neisseria spp, Streptococcus spp, S. mutans, Haemophilus spp., Moraxella spp, Bordetella spp, Mycobacterium spp, Legionella spp, Escherichia spp, Vibrio spp, Yersinia spp, Campylobacter spp, Salmonella spp, Listeria spp., Helicobacter spp, Pseudomonas spp, Staphylococcus spp., Enterococcus spp, Clostridium spp., Bacillus spp, Corynebacterium spp., Borrelia spp., Ehrlichia spp, Rickettsia spp, Chlamydia spp., Leptospira spp., Treponema spp.

In some embodiments, the anti-GITR antibodies, antibody fragments, or antigen binding molecules are co-administered with a tumor-associated antigen (TAA). The TAA can be an isolated polypeptide or peptide, can be part of an intact cell or part of a tumor cell lysate. Exemplary TAAs are discussed above; others known in the art also find use.

In some embodiments, the anti-GITR antibodies, antibody fragments, or antigen binding molecules are co-administered with autologous tumor cells from the patient, or allogeneic tumor cells of the same tissue type from another patient. The tumor cells can be in the form of intact cells, tumor cell lysate, apoptotic tumor cells or total tumor mRNA. The tumor cells can be transfected to express a polypeptide that enhances or augments the immunogenity of the tumor cell in the patient, e.g., transfected to express granulocyte colony stimulating factor (GM-CSF). The tumor cells can be from any cancerous tissue, including without limitation, epithelial cancers or carcinomas, as well as sarcomas and lymphomas. In some embodiments, the cancer is melanoma, ovarian cancer, renal cancer, colorectal cancer, prostate, lung cancer including non-small cell lung cancer (NSCLC), breast cancer, glioma, or fibrosarcoma. See, e.g., Pardee, et al, Immunotherapy (2009) 1(2):249-264, and references discussed therein. In one embodiment, the tumor cell is from, e.g., pancreatic cancer, melanomas, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues and head and neck squamous cell carcinoma (HNSCC).

Combinations with Immunomodulators

In certain embodiments, an immunomodulator used in the combinations with an anti GITR antibody molecule of the invention (e.g., in combination with a therapeutic agent chosen from an antigen-presentation combination) is an inhibitor of an immune checkpoint molecule. In one embodiment, the immunomodulator is an inhibitor of PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM (e.g., CEACAM-1, -3 and/or -5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFR beta. In one embodiment, the inhibitor of an immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3, CEACAM (e.g., CEACAM-1, -3 and/or -5), CTLA-4, or any combination thereof.

Inhibition of an inhibitory molecule can be performed at the DNA, RNA or protein level. In embodiments, an inhibitory nucleic acid (e.g., a dsRNA, siRNA or shRNA), can be used to inhibit expression of an inhibitory molecule. In other embodiments, the inhibitor of an inhibitory signal is, a polypeptide e.g., a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig), or an antibody or antigen-binding fragment thereof, that binds to the inhibitory molecule; e.g., an antibody or fragment thereof (also referred to herein as “an antibody molecule”) that binds to PD-1, PD-L1, PD-L2, CEACAM (e.g., CEACAM-1, -3 and/or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFR beta, or a combination thereof.

The GITR agonist can be administered alone, or in combination with other immunomodulators, e.g., in combination with an inhibitor of PD-1, PD-L1, CTLA-4, CEACAM (e.g., CEACAM-1, -3 and/or -5), TIM-3 or LAG-3. In one exemplary embodiment, the anti-GITR antibody molecule (e.g., as described herein) is administered in combination with an anti-PD-1 antibody molecule. The GITR antibody molecule and the anti-PD-1 antibody molecule may be in the form of separate antibody composition, or as a bispecific antibody molecule. In other embodiments, a GITR agonist can be administered in combination with other costimulatory molecule, e.g., an agonist of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand.

In certain embodiments, the antibody molecule is in the form of a bispecific or multispecific antibody molecule. In some embodiments, the anti-GITR antibody molecule is a bispecific antibody that binds to GITR and PD-1, PD-L1, CTLA-4, CEACAM (e.g., CEACAM-1, -3 and/or -5), TIM-3 or LAG-3. In one embodiment, the bispecific antibody molecule has a first binding specificity to GITR and a second binding specifity, e.g., a second binding specificity to PD-1, PD-L1 TIM-3, CEACAM (e.g., CEACAM-1, -3 and/or -5), LAG-3, or PD-L2.

In certain embodiments, the immunomodulator is an agonist of GITR, e.g., human GITR (e.g., an antibody molecule as described herein). In another embodiment, the immunomodulator is an antagonist, e.g., human GITRL. In one embodiment, the agonist of GITR and/or antagonist of GITRL is an antibody molecule to GITR. The GITR antibody can be administered alone, or in combination with other immunomodulators, e.g., in combination with an inhibitor of PD-1, PD-L1, LAG-3, TIM-3, CEACAM (e.g., CEACAM-1, -3 and/or -5) or CTLA-4. In an exemplary embodiment, the agonist of GITR, e.g., the anti-GITR antibody molecule, is administered in combination with a PD-1 or PD-L1 inhibitor. In another embodiment, the agonist of GITR, e.g., the anti-GITR antibody molecule, is administered in combination with a LAG-3 inhibitor, e.g., an anti-LAG-3 antibody molecule. In another embodiment, the agonist of GITR, e.g., the anti-GITR antibody molecule, is administered in combination with a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule. In another embodiment, the the agonist of GITR, e.g., the anti-GITR antibody molecule, is administered in combination with a CEACAM inhibitor (e.g., CEACAM-1, -3 and/or -5 inhibitor), e.g., an anti-CEACAM antibody molecule. In another embodiment, the the agonist of GITR, e.g., the anti-GITR antibody molecule, is administered in combination with a CEACAM-1 inhibitor, e.g., an anti-CEACAM-1 antibody molecule. In another embodiment, the the agonist of GITR, e.g., the anti-GITR antibody molecule, is administered in combination with a CEACAM-5 inhibitor, e.g., an anti-CEACAM-5 antibody molecule. In yet other embodiments, the agonist of GITR, e.g., the anti-GITR antibody molecule, is administered in combination with a LAG-3 inhibitor, e.g., an anti-LAG-3 antibody molecule, and a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule. Other combinations of immunomodulators with a GITR modulator (e.g., one or more of PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM (e.g., CEACAM-1, -3 and/or -5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFR) are also within the present invention. Any of the antibody molecules known in the art or disclosed herein can be used in the aforesaid combinations of inhibitors of checkpoint molecule.

In other embodiments, the immunomodulator is an inhibitor of CEACAM (e.g., CEACAM-1, -3 and/or -5), e.g., human CEACAM (e.g., CEACAM-1, -3 and/or -5). In one embodiment, the immunomodulator is an inhibitor of CEACAM-1, e.g., human CEACAM-1. In another embodiment, the immunomodulator is an inhibitor of CEACAM-3, e.g., human CEACAM-3. In another embodiment, the immunomodulator is an inhibitor of CEACAM-5, e.g., human CEACAM-5. In one embodiment, the inhibitor of CEACAM (e.g., CEACAM-1, -3 and/or -5) is an antibody molecule to CEACAM (e.g., CEACAM-1, -3 and/or -5). The CEACAM (e.g., CEACAM-1, -3 and/or -5) inhibitor can be administered alone, or in combination with other immunomodulators, e.g., in combination with an inhibitor of LAG-3, TIM-3, PD-1, PD-L1 or CTLA-4.

In other embodiments, the immunomodulator is an inhibitor of LAG-3, e.g., human LAG-3. In one embodiment, the inhibitor of LAG-3 is an antibody molecule to LAG-3. The LAG-3 inhibitor can be administered alone, or in combination with other immunomodulators, e.g., in combination with an inhibitor of CEACAM (e.g., CEACAM-1, -3 and/or -5), TIM-3, PD-1, PD-L1 or CTLA-4.

In other embodiments, the immunomodulator is an inhibitor of TIM-3, e.g., human TIM-3. In one embodiment, the inhibitor of TIM-3 is an antibody molecule to TIM-3. The TIM-3 inhibitor can be administered alone, or in combination with other immunomodulators, e.g., in combination with an inhibitor of CEACAM (e.g., CEACAM-1, -3 and/or -5), LAG-3, PD-1, PD-L1 or CTLA-4.

In certain embodiments, the immunomodulator used in the combinations disclosed herein (e.g., in combination with a therapeutic agent chosen from an antigen-presentation combination) is an activator or agonist of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is chosen from an agonist (e.g., an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion) of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand.

In other embodiments, the immunomodulator is an activator of a costimulatory molecule (e.g., an OX40 agonist). In one embodiment, the OX40 agonist is an antibody molecule to OX40. The OX40 agonist can be administered alone, or in combination with other immunomodulators, e.g., in combination with an inhibitor of PD-1, PD-L1, CTLA-4, CEACAM (e.g., CEACAM-1, -3 and/or -5), TIM-3 or LAG-3. In some embodiments, the anti-OX40 antibody molecule is a bispecific antibody that binds to GITR and PD-1, PD-L1, CTLA-4, CEACAM (e.g., CEACAM-1, -3 and/or -5), TIM-3 or LAG-3. In one exemplary embodiment, an OX40 antibody molecule is administered in combination with an anti-GITR antibody molecule (e.g., an anti-GITR molecule as described herein). The OX40 antibody molecule and the anti-GITR antibody molecule may be in the form of separate antibody composition, or as a bispecific antibody molecule. In other embodiments, the OX40 agonist can be administered in combination with other costimulatory molecule, e.g., an agonist of GITR, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand.

In other embodiments, the immunomodulator is an activator of a costimulatory molecule (e.g., an 4-1BB (CD-137) agonist). In one embodiment, the 4-1BB (CD-137) agonist is an antibody molecule to 4-1BB (CD-137). In some embodiments, the combination disclosed herein, e.g., a combination comprising an anti-GITR antibody molecule, is administered with a 4-1BB receptor targeting agent (e.g., an antibody that stimulates signaling through 4-1BB (CD-137), e.g., PF-2566). In one embodiment, the anti-GITR antibody molecule is administered in combination with a tyrosine kinase inhibitor (e.g., axitinib) and a 4-1BB receptor targeting agent.

The 4-1BB agonist can be combined alone, or in combination with other immunomodulators, e.g., in combination with an inhibitor of PD-1, PD-L1, CTLA-4, CEACAM (e.g., CEACAM-1, -3 and/or -5), TIM-3 or LAG-3. In some embodiments, the anti-4-1BB antibody molecule is a bispecific antibody that binds to GITR and PD-1, PD-L1, CTLA-4, CEACAM (e.g., CEACAM-1, -3 and/or -5), TIM-3 or LAG-3. In one exemplary embodiment, an 4-1BB antibody molecule is administered in combination with an anti-GITR antibody molecule (e.g., an anti-GITR molecule as described herein). The 4-1BB antibody molecule and the anti-GITR antibody molecule may be in the form of separate antibody composition, or as a bispecific antibody molecule. In other embodiments, the 4-1BB agonist can be administered in combination with other costimulatory molecule, e.g., an agonist of GITR, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand.

It is noted that only exemplary combinations of inhibitors of checkpoint inhibitors or agonists of costimulatory molecules are provided herein. Additional combinations of these agents are within the scope of the present invention.

In some embodiments, the anti-GITR antibodies, antibody fragments, or antigen binding molecules are co-administered with a cytotoxic agent. For example, the anti-GITR antibodies or antigen binding molecules are co-administered with an agonist antibody or antigen binding molecule that binds to and reduces or depletes CD4+CD25+ regulatory T cells (Treg). Exemplary Treg cell-depleting antibodies or antigen binding molecules bind to CD25 or CCR4. See, Expert Opin Ther Patents (2007) 17(5):567-575, and the references discussed therein.

In some embodiments, the anti-GITR antibodies, antibody fragments, or antigen binding molecules are co-administered with an inhibitor of a co-inhibitory signal. Exemplary inhibitors include inhibitors of CTLA-4, LAG3, TIM3 and/or inhibitors of the PD-1/PD-L1 (e.g., B7-H1) interaction. In some embodiments, the anti-GITR antibodies are co-administered with an antibody that binds to and inhibits CTLA-4. In some embodiments, the anti-GITR antibodies are co-administered with an antibody that binds to and inhibits TIM3. In some embodiments, the anti-GITR antibodies are co-administered with an antibody that binds to and inhibits LAG3. In some embodiments, the anti-GITR antibodies are co-administered with an antibody that binds to and inhibits PD-1. In some embodiments, the anti-GITR antibodies are co-administered with an antibody that binds to and inhibits B7-H1. See, e.g., Expert Opin Ther Patents (2007) 17(5):567-575; and Melero, et al., Clin Cancer Res (2009) 15(5):1507-1509, and the references discussed therein.

The anti-GITR antibodies, antibody fragments, or antigen binding molecules can also be co-administered with one or more immunostimulatory agents. For example, in some embodiments, the anti-GITR antibodies or antibody fragments are co-administered with an immunostimulatory cytokine, for example, IL-7, IL-12 or IL-15. Alternatively, the anti-GITR antibodies, antibody fragments, or antigen binding molecules can be co-administered with a second immunostimulatory antibody. For example, the anti-GITR antibodies, antibody fragments, or antigen binding molecules can also be co-administered with an agonist antibody, antibody fragment, or antigen binding molecule of another member of the tumor necrosis factor receptor superfamily. Exemplary secondary immunostimulatory targets include without limitation TNFRSF4 tumor necrosis factor receptor superfamily, member 4 (also known as OX40) or tumor necrosis factor receptor superfamily, member 9 (also known as TNFRSF9, 4-1BB or CD137). See, e.g., Expert Opin Ther Patents (2007) 17(5):567-575; Pardee, et al, Immunotherapy (2009) 1(2):249-264; and Melero, et al., Clin Cancer Res (2009) 15(5):1507-1509, and the references discussed therein.

The anti-GITR antibodies, antibody fragments, or antigen binding molecules can also be co-administered with a chemotherapeutic agent. The selected agent will depend on the condition to be treated, e.g., a cancer or an infectious disease, such as a bacterial infection, a fungal infection, a viral infection or a parasitic infection. The anti-GITR antibodies, antibody fragments, or antigen binding molecules can be co-administered with a chemotherapeutic known by those of skill to treat the disease condition being treated. Exemplary chemotherapeutic agents are discussed above; others known in the art also find use.

Additional Combination Therapies

Combinations disclosed herein, e.g., combination of PD-1 antibody molecules, with one or more further therapeutics are provided herein. Many of the combinations in this section are useful in treating cancer, but other indications are also described. This section focuses on combinations of anti-GITR-1 antibody molecules of the invention, optionally in combination with one or more immunomodulators (e.g., an anti-PD-1 antibody molecule, an anti-TIM-3 antibody molecule, an anti-LAG-3 antibody molecule, or an anti-PD-L1 antibody molecule), with one or more of the agents described in Table 6. In the combinations herein below, in one embodiment, the anti-GITR antibody molecule includes: an antibody or an antibody fragment, or antigen binding molecule thereof that binds to SEQ ID NO:1, and wherein the antibody, antibody fragment, or antigen binding molecule comprises

-   -   (a) a heavy chain variable region, wherein:         -   i) the heavy chain CDR1 comprises SEQ ID NO:22, and         -   ii) the heavy chain CDR2 comprises a sequence selected from             any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID             NO:26, and SEQ ID NO:27, and         -   ii) the heavy chain CDR3 comprises SEQ ID NO:29 or SEQ ID             NO:109; and     -   (b) a light chain variable region, wherein         -   i) the light chain CDR1 comprises SEQ ID NO:30 or SEQ ID             NO:31, and         -   ii) the light chain CDR2 comprises SEQ ID NO:33, and         -   iii) the light chain CDR3 comprises SEQ ID NO:34.

Combination Therapies

In certain embodiments, any of the combinations disclosed herein further includes one or more of the agents described in Table 6.

In some embodiments, the additional therapeutic agent is chosen from one or more of: 1) a protein kinase C (PKC) inhibitor; 2) a heat shock protein 90 (HSP90) inhibitor; 3) an inhibitor of a phosphoinositide 3-kinase (PI3K) and/or target of rapamycin (mTOR); 4) an inhibitor of cytochrome P450 (e.g., a CYP17 inhibitor or a 17alpha-Hydroxylase/C17-20 Lyase inhibitor); 5) an iron chelating agent; 6) an aromatase inhibitor; 7) an inhibitor of p53, e.g., an inhibitor of a p53/Mdm2 interaction; 8) an apoptosis inducer; 9) an angiogenesis inhibitor; 10) an aldosterone synthase inhibitor; 11) a smoothened (SMO) receptor inhibitor; 12) a prolactin receptor (PRLR) inhibitor; 13) a Wnt signaling inhibitor; 14) a CDK4/6 inhibitor; 15) a fibroblast growth factor receptor 2 (FGFR2)/fibroblast growth factor receptor 4 (FGFR4) inhibitor; 16) an inhibitor of macrophage colony-stimulating factor (M-CSF); 17) an inhibitor of one or more of c-KIT, histamine release, Flt3 (e.g., FLK2/STK1) or PKC; 18) an inhibitor of one or more of VEGFR-2 (e.g., FLK-1/KDR), PDGFRbeta, c-KIT or Raf kinase C; 19) a somatostatin agonist and/or a growth hormone release inhibitor; 20) an anaplastic lymphoma kinase (ALK) inhibitor; 21) an insulin-like growth factor 1 receptor (IGF-1R) inhibitor; 22) a P-Glycoprotein 1 inhibitor; 23) a vascular endothelial growth factor receptor (VEGFR) inhibitor; 24) a BCR-ABL kinase inhibitor; 25) an FGFR inhibitor; 26) an inhibitor of CYP11B2; 27) a HDM2 inhibitor, e.g., an inhibitor of the HDM2-p53 interaction; 28) an inhibitor of a tyrosine kinase; 29) an inhibitor of c-MET; 30) an inhibitor of JAK; 31) an inhibitor of DAC; 32) an inhibitor of 11β-hydroxylase; 33) an inhibitor of IAP; 34) an inhibitor of PIM kinase; 35) an inhibitor of Porcupine; 36) an inhibitor of BRAF, e.g., BRAF V600E or wild-type BRAF; 37) an inhibitor of HER3; 38) an inhibitor of MEK; or 39) an inhibitor of a lipid kinase, e.g., as described herein and in Table 6.

In one embodiment, the additional therapeutic agent is chosen from one or more of: Compound A8, Compound A17, Compound A23, Compound A24, Compound A27, Compound A29, Compound A33, and Compound A13.

In other embodiments, the additional therapeutic agent is chosen from one or more of: Compound A5, Compound A8, Compound A17, Compound A23, Compound A24, Compound A29, and Compound A40.

In other embodiments, the additional therapeutic agent is chosen from one or more of: Compound A9, Compound A16, Compound A17, Compound A21, Compound A22, Compound A25, Compound A28, Compound A48, and Compound 49.

In one embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a PKC inhibitor, Sotrastaurin (Compound A1), or a compound disclosed in PCT Publication No. WO 2005/039549, to treat a disorder, e.g., a disorder described herein. In one embodiment, the PKC inhibitor is Sotrastaurin (Compound A1) or a compound disclosed in PCT Publication No. WO 2005/039549. In one embodiment, a GITR antibody molecule is used in combination with Sotrastaurin (Compound A1), or a compound as described in PCT Publication No. WO 2005/039549, to treat a disorder such as a cancer, a melanoma, a non-Hodgkin lymphoma, an inflammatory bowel disease, transplant rejection, an ophthalmic disorder, or psoriasis.

In certain embodiments, Sotrastaurin (Compound A1) is administered at a dose of about 20 to 600 mg, e.g., about 200 to about 600 mg, about 50 mg to about 450 mg, about 100 mg to 400 mg, about 150 mg to 350 mg, or about 200 mg to 300 mg, e.g., about 50 mg, 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg. The dosing schedule can vary from e.g., every other day to daily, twice or three times a day.

In one embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as provided herein, is used in combination with a BCR-ABL inhibitor, TASIGNA (Compound A2), or a compound disclosed in PCT Publication No. WO 2004/005281, to treat a disorder, e.g., a disorder described herein. In one embodiment, the BCR-ABL inhibitor is TASIGNA, or a compound disclosed in PCT Publication No. WO 2004/005281. In one embodiment, a GITR antibody molecule is used in combination with TASIGNA (Compound A2), or a compound as described in PCT Publication No. WO 2004/005281, to treat a disorder such as a lymphocytic leukemia, Parkinson's Disease, a neurologic cancer, a melanoma, a digestive/gastrointestinal cancer, a colorectal cancer, a myeloid leukemia, a head and neck cancer, or pulmonary hypertension.

In one embodiment, the BCR-ABL inhibitor or TASIGNA is administered at a dose of about 300 mg (e.g., twice daily, e.g., for newly diagnosed Ph+ CML-CP), or about 400 mg, e.g., twice daily, e.g., for resistant or intolerant Ph+ CML-CP and CML-AP). BCR-ABL inhibitor or a Compound A2 is administered at a dose of about 300-400 mg.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as provided herein, is used in combination with an HSP90 inhibitor, such as 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (Compound A3), or a compound disclosed in PCT Publication No. WO 2010/060937 or WO 2004/072051, to treat a disorder, e.g., a disorder described herein. In one embodiment, the HSP90 inhibitor is 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (Compound A3), or a compound disclosed in PCT Publication No. WO 2010/060937 or WO 2004/072051. In one embodiment, a GITR antibody molecule is used in combination with 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (Compound A3), or a compound as described in PCT Publication No. WO 2010/060937 or WO 2004/072051, to treat a disorder such as a cancer, a multiple myeloma, a non-small cell lung cancer, a lymphoma, a gastric cancer, a breast cancer, a digestive/gastrointestinal cancer, a pancreatic cancer, a colorectal cancer, a solid tumor, or a hematopoiesis disorder.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an inhibitor of PI3K and/or mTOR, Dactolisib (Compound A4) or 8-(6-Methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (Compound A41), or a compound disclosed in PCT Publication No. WO 2006/122806, to treat a disorder, e.g., a disorder described herein. In one embodiment, the PI3K and/or mTOR inhibitor is Dactolisib (Compound A4), 8-(6-Methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (Compound A41), or a compound disclosed in PCT Publication No. WO 2006/122806. In one embodiment, a GITR antibody molecule is used in combination with Dactolisib (Compound A4), 8-(6-Methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (Compound A41), or a compound described in PCT Publication No. WO 2006/122806, to treat a disorder such as a cancer, a prostate cancer, a leukemia (e.g., lymphocytic leukemia), a breast cancer, a brain cancer, a bladder cancer, a pancreatic cancer, a renal cancer, a solid tumor, or a liver cancer.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an FGFR inhibitor, 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6-((4-(4-ethylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methylurea (Compound A5) or a compound disclosed in U.S. Pat. No. 8,552,002, to treat a disorder, e.g., a disorder described herein. In one embodiment, the FGFR inhibitor is 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6-((4-(4-ethylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methylurea (Compound A5) or a compound disclosed in U.S. Pat. No. 8,552,002. In one embodiment, a GITR antibody molecule is used in combination with Compound A5, or a compound as described in U.S. Pat. No. 8,552,002, to treat a disorder such as a digestive/gastrointestinal cancer, a hematological cancer, or a solid tumor.

In one embodiment, the FGFR inhibitor or 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6-((4-(4-ethylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methylurea (Compound A5) is administered at a dose of about 100-125 mg (e.g., per day), e.g., about 100 mg or about 125 mg.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a PI3K inhibitor, Buparlisib (Compound A6), or a compound disclosed in PCT Publication No. WO 2007/084786, to treat a disorder, e.g., a disorder described herein. In one embodiment, the PI3K inhibitor is Buparlisib (Compound A6) or a compound disclosed in PCT Publication No. WO 2007/084786. In one embodiment, a GITR antibody molecule is used in combination with Buparlisib (Compound A6), or a compound disclosed in PCT Publication No. WO 2007/084786, to treat a disorder such as, a prostate cancer, a non-small cell lung cancer, an endocrine cancer, a leukemia, an ovarian cancer, a melanoma, a bladder cancer, a breast cancer, a female reproductive system cancer, a digestive/gastrointestinal cancer, a colorectal cancer, a glioblastoma multiforme, a solid tumor, a non-Hodgkin lymphoma, a hematopoiesis disorder, or a head and neck cancer.

In one embodiment, the PI3K inhibitor or Buparlisib (Compound A6) is administered at a dose of about 100 mg (e.g., per day).

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an FGFR inhibitor, 8-(2,6-difluoro-3,5-dimethoxyphenyl)-N-(4-((dimethylamino)methyl)-1H-imidazol-2-yl)quinoxaline-5-carboxamide (Compound A7) or a compound disclosed in PCT Publication No. WO 2009/141386 to treat a disorder, e.g., a disorder described herein. In one embodiment, the FGFR inhibitor is 8-(2,6-difluoro-3,5-dimethoxyphenyl)-N-(4-((dimethylamino)methyl)-1H-imidazol-2-yl)quinoxaline-5-carboxamide(Compound A7) or a compound disclosed in a PCT Publication No. WO 2009/141386. In one embodiment, the FGFR inhibitor is 8-(2,6-difluoro-3,5-dimethoxyphenyl)-N-(4-((dimethylamino)methyl)-1H-imidazol-2-yl)quinoxaline-5-carboxamide(Compound A7). In one embodiment, a GITR antibody molecule is used in combination with 8-(2,6-difluoro-3,5-dimethoxyphenyl)-N-(4-((dimethylamino)methyl)-1H-imidazol-2-yl)quinoxaline-5-carboxamide(Compound A7), or a compound disclosed in PCT Publication No. WO 2009/141386, to treat a disorder such as a cancer characterized by angiogenesis.

In one embodiment, the FGFR inhibitor or 8-(2,6-difluoro-3,5-dimethoxyphenyl)-N-(4-((dimethylamino)methyl)-1H-imidazol-2-yl)quinoxaline-5-carboxamide (Compound A7) is administered at a dose of e.g., from approximately 3 mg to approximately 5 g, more preferably from approximately 10 mg to approximately 1.5 g per person per day, optionally divided into 1 to 3 single doses which may, for example, be of the same size.

In another embodiment the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a PI3K inhibitor, (S)—N1-(4-methyl-5-(2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl)thiazol-2-yl)pyrrolidine-1,2-dicarboxamide (Compound A8) or a compound disclosed PCT Publication No. WO 2010/029082 to treat a disorder, e.g., a disorder described herein. In one embodiment, the PI3K inhibitor is (S)—N1-(4-methyl-5-(2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl)thiazol-2-yl)pyrrolidine-1,2-dicarboxamide (Compound A8) or a compound disclosed PCT Publication No. WO 2010/029082. In one embodiment, a GITR antibody molecule is used in combination with (S)—N1-(4-methyl-5-(2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl)thiazol-2-yl)pyrrolidine-1,2-dicarboxamide (Compound A8), or a compound disclosed PCT Publication No. WO 2010/029082, to treat a disorder such as a gastric cancer, a breast cancer, a pancreatic cancer, a digestive/gastrointestinal cancer, a solid tumor, and a head and neck cancer.

In one embodiment, the PI3K inhibitor or (S)—N1-(4-methyl-5-(2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl)thiazol-2-yl)pyrrolidine-1,2-dicarboxamide (Compound A8) is administered at a dose of about 150-300, 200-300, 200-400, or 300-400 mg (e.g., per day), e.g., about 200, 300, or 400 mg.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an inhibitor of cytochrome P450 (e.g., a CYP17 inhibitor) or a compound disclosed in PCT Publication No. WO 2010/149755, to treat a disorder, e.g., a disorder described herein. In one embodiment, the cytochrome P450 inhibitor (e.g., the CYP17 inhibitor) is a compound disclosed in PCT Publication No. WO 2010/149755. In one embodiment, a GITR antibody molecule is used in combination with a compound disclosed in PCT Publication No. WO 2010/149755, to treat prostate cancer.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an HDM2 inhibitor, (S)-1-(4-chlorophenyl)-7-isopropoxy-6-methoxy-2-(4-(methyl(((1r,4S)-4-(4-methyl-3-oxopiperazin-1-yl)cyclohexyl)methyl)amino)phenyl)-1,2-dihydroisoquinolin-3(4H)-one(Compound A10) or a compound disclosed in PCT Publication No. WO 2011/076786 to treat a disorder, e.g., a disorder described herein). In one embodiment, the HDM2 inhibitor is (S)-1-(4-chlorophenyl)-7-isopropoxy-6-methoxy-2-(4-(methyl(((1r,4S)-4-(4-methyl-3-oxopiperazin-1-yl)cyclohexyl)methyl)amino)phenyl)-1,2-dihydroisoquinolin-3(4H)-one (Compound A10) or a compound disclosed in PCT Publication No. WO 2011/076786. In one embodiment, a GITR antibody molecule is used in combination with (S)-1-(4-chlorophenyl)-7-isopropoxy-6-methoxy-2-(4-(methyl(((1r,4S)-4-(4-methyl-3-oxopiperazin-1-yl)cyclohexyl)methyl)amino)phenyl)-1,2-dihydroisoquinolin-3(4H)-one (Compound A10), or a compound disclosed in PCTPublication No. WO 2011/076786, to treat a disorder such as a solid tumor.

In one embodiment, the HDM2 inhibitor or (S)-1-(4-chlorophenyl)-7-isopropoxy-6-methoxy-2-(4-(methyl(((1r,4S)-4-(4-methyl-3-oxopiperazin-1-yl)cyclohexyl)methyl)amino)phenyl)-1,2-dihydroisoquinolin-3(4H)-one (Compound A10) is administered at a dose of about 400 to 700 mg, e.g., administered three times weekly, 2 weeks on and one week off. In some embodiments, the dose is about 400, 500, 600, or 700 mg; about 400-500, 500-600, or 600-700 mg, e.g., administered three times weekly.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an iron chelating agent, Deferasirox (also known as EXJADE; Compound A11), or a compound disclosed in PCT Publication No. WO 1997/049395 to treat a disorder, e.g., a disorder described herein. In one embodiment, the iron chelating agent is Deferasirox or a compound disclosed in PCT Publication No. WO 1997/049395. In one embodiment, the iron chelating agent is Deferasirox (Compound A11). In one embodiment, a GITR antibody molecule is used in combination with Deferasirox (Compound A11), or a compound disclosed in PCT Publication No. WO 1997/049395, to treat iron overload, hemochromatosis, or myelodysplasia.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an aromatase inhibitor, Letrozole (also known as FEMARA; Compound A12), or a compound disclosed in U.S. Pat. No. 4,978,672 to treat a disorder, e.g., a disorder described herein. In one embodiment, the aromatase inhibitor is Letrozole (Compound A12) or a compound disclosed in U.S. Pat. No. 4,978,672. In one embodiment, a GITR antibody molecule is used in combination with Letrozole (Compound A12), or a compound disclosed in U.S. Pat. No. 4,978,672, to treat a disorder such as a cancer, a leiomyosarcoma, an endometrium cancer, a breast cancer, a female reproductive system cancer, or a hormone deficiency.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a PI3K inhibitor, e.g., a pan-PI3K inhibitor, (4S,5R)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-4-(hydroxymethyl)-5-methyloxazolidin-2-one (Compound A13) or a compound disclosed in PCT Publication No. WO2013/124826 to treat a disorder, e.g., a disorder described herein. In one embodiment, the PI3K inhibitor is (4S,5R)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-4-(hydroxymethyl)-5-methyloxazolidin-2-one (Compound A13) or a compound disclosed in PCT Publication No. WO2013/124826. In one embodiment, a GITR antibody molecule is used in combination with (4S,5R)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-4-(hydroxymethyl)-5-methyloxazolidin-2-one (Compound A13), or a compound disclosed in PCT Publication No. WO2013/124826, to treat a disorder such as a cancer or an advanced solid tumor.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an inhibitor of p53 and/or a p53/Mdm2 interaction, (S)-5-(5-chloro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-1-isopropyl-5,6-dihydropyrrolo[3,4-d]imidazol-4(1H)-one (Compound A14), or a compound disclosed in PCT Publication No.

WO2013/111105 to treat a disorder, e.g., a disorder described herein. In one embodiment, the p53 and/or a p53/Mdm2 interaction inhibitor is (S)-5-(5-chloro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-1-isopropyl-5,6-dihydropyrrolo[3,4-d]imidazol-4(1H)-one (Compound A14) or a compound disclosed in PCT Publication No. WO2013/111105. In one embodiment, a GITR antibody molecule is used in combination with (S)-5-(5-chloro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-1-isopropyl-5,6-dihydropyrrolo[3,4-d]imidazol-4(1H)-one (Compound A14), or a compound disclosed in PCT Publication No. WO2013/111105, to treat a disorder such as a cancer or a soft tissue sarcoma.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a CSF-1R tyrosine kinase inhibitor, 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide (Compound A15), or a compound disclosed in PCT Publication No. WO 2005/073224 to treat a disorder, e.g., a disorder described herein. In one embodiment, the CSF-1R tyrosine kinase inhibitor is 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide (Compound A15) or a compound disclosed in PCT Publication No. WO 2005/073224. In one embodiment, a GITR antibody molecule is used in combination with 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide (Compound A15) or a compound disclosed in PCT Publication No. WO 2005/073224, to treat a disorder such as cancer.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an apoptosis inducer and/or an angiogenesis inhibitor, such as Imatinib mesylate (also known as GLEEVEC; Compound A16) or a compound disclosed in PCT Publication No. WO1999/003854 to treat a disorder, e.g., a disorder described. In one embodiment, the apoptosis inducer and/or an angiogenesis inhibitor is Imatinib mesylate (Compound A16) or a compound disclosed in PCT Publication No. WO1999/003854. In one embodiment, a GITR antibody molecule is used in combination with Imatinib mesylate (Compound A16), or a compound disclosed in PCT Publication No. WO1999/003854, to treat a disorder such as a cancer, a multiple myeloma, a prostate cancer, a non-small cell lung cancer, a lymphoma, a gastric cancer, a melanoma, a breast cancer, a pancreatic cancer, a digestive/gastrointestinal cancer, a colorectal cancer, a glioblastoma multiforme, a liver cancer, a head and neck cancer, asthma, multiple sclerosis, allergy, Alzheimer's dementia, amyotrophic lateral sclerosis, or rheumatoid arthritis.

In certain embodiments, Imatinib mesylate (Compound A16) is administered at a dose of about 100 to 1000 mg, e.g., about 200 mg to 800 mg, about 300 mg to 700 mg, or about 400 mg to 600 mg, e.g., about 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, or 700 mg. The dosing schedule can vary from e.g., every other day to daily, twice or three times a day. In one embodiment, Imatinib mesylate is administered at an oral dose from about 100 mg to 600 mg daily, e.g., about 100 mg, 200 mg, 260 mg, 300 mg, 400 mg, or 600 mg daily.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a JAK inhibitor, 2-fluoro-N-methyl-4-(7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl)benzamide (Compound A17), or a dihydrochloric salt thereof, or a compound disclosed in PCT Publication No. WO 2007/070514, to treat a disorder, e.g., a disorder described herein. In one embodiment, the JAK inhibitor is 2-fluoro-N-methyl-4-(7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl)benzamide (Compound A17), or a dihydrochloric salt thereof, or a compound disclosed in PCT Publication No. WO 2007/070514. In one embodiment, a GITR antibody molecule is used in combination with 2-fluoro-N-methyl-4-(7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl)benzamide (Compound A17), or a dihydrochloric salt thereof, or a compound disclosed in PCT Publication No. WO 2007/070514, to treat a disorder such as colorectal cancer, myeloid leukemia, hematological cancer, autoimmune disease, non-Hodgkin lymphoma, or thrombocythemia.

In one embodiment, the JAK inhibitor or a 2-fluoro-N-methyl-4-(7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl)benzamide (Compound A17), or a dihydrochloric salt thereof is administered at a dose of about 400-600 mg (e.g., per day), e.g., about 400, 500, or 600 mg, or about 400-500 or 500-600 mg.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a JAK inhibitor, Ruxolitinib Phosphate (also known as JAKAFI; Compound A18) or a compound disclosed in PCT Publication No. WO 2007/070514 to treat a disorder, e.g., a disorder described herein. In one embodiment, the JAK inhibitor is Ruxolitinib Phosphate (Compound A18) or a compound disclosed in PCT Publication No. WO 2007/070514. In one embodiment, a GITR antibody molecule is used in combination with Ruxolitinib Phosphate (Compound A18), or a compound disclosed in PCT Publication No. WO 2007/070514, to treat a disorder such as a prostate cancer, a lymphocytic leukemia, a multiple myeloma, a lymphoma, a lung cancer, a leukemia, cachexia, a breast cancer, a pancreatic cancer, rheumatoid arthritis, psoriasis, a colorectal cancer, a myeloid leukemia, a hematological cancer, an autoimmune disease, a non-Hodgkin lymphoma, or thrombocythemia.

In one embodiment, the JAK inhibitor or Ruxolitinib Phosphate (Compound A18) is administered at a dose of about 15-25 mg, e.g., twice daily. In some embodiments, the dose is about 15, 20, or 25 mg, or about 15-20 or 20-25 mg.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a deacetylase (DAC) inhibitor, Panobinostat (Compound A19), or a compound disclosed in PCT Publication No. WO 2014/072493 to treat a disorder, e.g., a disorder described herein. In one embodiment, the DAC inhibitor is Panobinostat (Compound A19) or a compound disclosed in PCT Publication No. WO 2014/072493. In one embodiment, a GITR antibody molecule is used in combination with Panobinostat (Compound A19), a compound disclosed in PCT Publication No. WO 2014/072493, to treat a disorder such as a small cell lung cancer, a respiratory/thoracic cancer, a prostate cancer, a multiple myeloma, myelodysplastic syndrome, a bone cancer, a non-small cell lung cancer, an endocrine cancer, a lymphoma, a neurologic cancer, a leukemia, HIV/AIDS, an immune disorder, transplant rejection, a gastric cancer, a melanoma, a breast cancer, a pancreatic cancer, a colorectal cancer, a glioblastoma multiforme, a myeloid leukemia, a hematological cancer, a renal cancer, a non-Hodgkin lymphoma, a head and neck cancer, a hematopoiesis disorders, or a liver cancer.

In one embodiment, the DAC inhibitor or Panobinostat (Compound A19) is administered at a dose of about 20 mg (e.g., per day).

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an inhibitor of one or more of cytochrome P450 (e.g., 11B2), aldosterone or angiogenesis, Osilodrostat (Compound A20), or a compound disclosed in PCT Publication No. WO2007/024945 to treat a disorder, e.g., a disorder described herein. In one embodiment, the inhibitor of one or more of cytochrome P450 (e.g., 11B2), aldosterone or angiogenesis is Osilodrostat (Compound A20) or a compound disclosed in PCT Publication No. WO2007/024945. In one embodiment, a GITR antibody molecule is used in combination with Osilodrostat (Compound A20), or a compound disclosed in PCT Publication No. WO2007/024945, to treat a disorder such as Cushing's syndrome, hypertension, or heart failure therapy.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a IAP inhibitor, (S)—N—((S)-1-cyclohexyl-2-((S)-2-(4-(4-fluorobenzoyl)thiazol-2-yl)pyrrolidin-1-yl)-2-oxoethyl)-2-(methylamino)propanamide (Compound A21) or a compound disclosed in U.S. Pat. No. 8,552,003 to treat a disorder, e.g., a disorder described herein. In one embodiment, the IAP inhibitor is (S)—N—((S)-1-cyclohexyl-2-((S)-2-(4-(4-fluorobenzoyl)thiazol-2-yl)pyrrolidin-1-yl)-2-oxoethyl)-2-(methylamino)propanamide (Compound A21) or a compound disclosed in U.S. Pat. No. 8,552,003. In one embodiment, a GITR antibody molecule is used in combination with (S)—N—((S)-1-cyclohexyl-2-((S)-2-(4-(4-fluorobenzoyl)thiazol-2-yl)pyrrolidin-1-yl)-2-oxoethyl)-2-(methylamino)propanamide (Compound A21), or a compound disclosed in U.S. Pat. No. 8,552,003, to treat a disorder such as a multiple myeloma, a breast cancer, an ovarian cancer, a pancreatic cancer, or a hematopoiesis disorder.

In one embodiment, the IAP inhibitor or (S)—N—((S)-1-cyclohexyl-2-((S)-2-(4-(4-fluorobenzoyl)thiazol-2-yl)pyrrolidin-1-yl)-2-oxoethyl)-2-(methylamino)propanamide (Compound A21) or a compound disclosed in U.S. Pat. No. 8,552,003 is administered at a dose of approximately 1800 mg, e.g., once weekly.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination a Smoothened (SMO) inhibitor, Sonidegib phosphate (Compound A22), (R)-2-(5-(4-(6-benzyl-4,5-dimethylpyridazin-3-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)propan-2-ol (Compound A25), or a compound disclosed in PCT Publication No. WO 2007/131201 or WO 2010/007120 to treat a disorder, e.g., a disorder described herein. In one embodiment, the SMO inhibitor is Sonidegib phosphate (Compound A22), (R)-2-(5-(4-(6-benzyl-4,5-dimethylpyridazin-3-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)propan-2-ol (Compound A25), or a compound disclosed in PCT Publication No. WO 2007/131201 or WO 2010/007120. In one embodiment, a GITR antibody molecule is used in combination with Sonidegib phosphate (Compound A22), (R)-2-(5-(4-(6-benzyl-4,5-dimethylpyridazin-3-yl)-2-methylpiperazin-1-yl)pyrazin-2-yl)propan-2-ol (Compound A25), or a compound disclosed in PCT Publication No. WO 2007/131201 or WO 2010/007120 to treat a disorder such as a cancer, a medulloblastoma, a small cell lung cancer, a prostate cancer, a basal cell carcinoma, a pancreatic cancer, or an inflammation.

In certain embodiments, Sonidegib phosphate (Compound A22) is administered at a dose of about 20 to 500 mg, e.g., about 40 mg to 400 mg, about 50 mg to 300 mg, or about 100 mg to 200 mg, e.g., about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, or 300 mg. The dosing schedule can vary from e.g., every other day to daily, twice or three times a day.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an Alk inhibitor, ceritinib (also known as ZYKADIA; Compound A23) or a compound disclosed in PCT Publication No. WO 2007/131201 to treat a disorder, e.g., a disorder described herein. In one embodiment, the Alk inhibitor is ceritinib (Compound A23) or a compound disclosed in PCT Publication No. WO 2007/131201. In one embodiment, a GITR antibody molecule is used in combination with ceritinib (Compound A23), or a compound disclosed in PCT Publication No. WO 2007/131201, to treat a disorder such as non-small cell lung cancer or solid tumors.

In one embodiment, the Alk inhibitor or ceritinib (Compound A23) is administered at a dose of approximately 750 mg, e.g., once daily.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a JAK and/or CDK4/6 inhibitor, 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound A24), or a compound disclosed in U.S. Pat. No. 8,415,355 or U.S. Pat. No. 8,685,980 to treat a disorder, e.g., a disorder described herein. In one embodiment, the JAK and/or CDK4/6 inhibitor is 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound A24) or a compound disclosed in U.S. Pat. No. 8,415,355 or U.S. Pat. No. 8,685,980. In one embodiment, a GITR antibody molecule is used in combination with 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound A24), or a compound disclosed in U.S. Pat. No. 8,415,355 or U.S. Pat. No. 8,685,980, to treat a disorder such as a lymphoma, a neurologic cancer, a melanoma, a breast cancer, or a solid tumor.

In one embodiment, the JAK and/or CDK4/6 inhibitor or 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound A24) is administered at a dose of approximately 200-600 mg, e.g., per day. In one embodiment, the compound is administered at a dose of about 200, 300, 400, 500, or 600 mg, or about 200-300, 300-400, 400-500, or 500-600 mg.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination a prolactin receptor (PRLR) inhibitor, a human monoclonal antibody molecule (Compound A26) as disclosed in U.S. Pat. No. 7,867,493), to treat a disorder, e.g., a disorder described herein. In one embodiment, the PRLR inhibitor is a human monoclonal antibody (Compound A26) disclosed in U.S. Pat. No. 7,867,493. In one embodiment, a GITR antibody molecule is used in combination with human monoclonal antibody molecule (Compound A26) described in U.S. Pat. No. 7,867,493 to treat a disorder such as, a cancer, a prostate cancer, or a breast cancer.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a PIM Kinase inhibitor, N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide (Compound A27) or a compound disclosed in PCT Publication No. WO 2010/026124 to treat a disorder, e.g., a disorder described herein. In one embodiment, the PIM Kinase inhibitor is N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide (Compound A27) or a compound disclosed in PCT Publication No. WO 2010/026124. In one embodiment, a GITR antibody molecule is used in combination with N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide (Compound A27), or a compound disclosed in PCT Publication No. WO 2010/026124, to treat a disorder such as a multiple myeloma, myelodysplastic syndrome, a myeloid leukemia, or a non-Hodgkin lymphoma.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination a Wnt signaling inhibitor, 2-(2′,3-dimethyl-[2,4′-bipyridin]-5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (Compound A28) or a compound disclosed in PCT publication No. WO 2010/101849 to treat a disorder, e.g., a disorder described herein. In one embodiment, the Wnt signaling inhibitor is 2-(2′,3-dimethyl-[2,4′-bipyridin]-5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (Compound A28) or a compound disclosed in PCT publication No. WO 2010/101849. In one embodiment, the Wnt signaling inhibitor is 2-(2′,3-dimethyl-[2,4′-bipyridin]-5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (Compound A28). In one embodiment, a GITR antibody molecule is used in combination with 2-(2′,3-dimethyl-[2,4′-bipyridin]-5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (Compound A28), or a compound disclosed in PCT publication No. WO 2010/101849, to treat a disorder such as a solid tumor (e.g., a head and neck cancer, a squamous cell carcinoma, a breast cancer, a pancreatic cancer, or a colon cancer).

In certain embodiments, 2-(2′,3-dimethyl-[2,4′-bipyridin]-5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (Compound A28) is administered at a dose of about 1 to 50 mg, e.g., about 2 mg to 45 mg, about 3 mg to 40 mg, about 5 mg to 35 mg, 5 mg to 10 mg, or about 10 mg to 30 mg, e.g., about 2 mg, 5 mg, 10 mg, 20 mg, 30 mg, or 40 mg. The dosing schedule can vary from e.g., every other day to daily, twice or three times a day.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a BRAF inhibitor, Encorafenib (Compound A29), or a compound disclosed in PCT Publication No. WO 2011/025927 to treat a disorder, e.g., a disorder described herein. In one embodiment, the BRAF inhibitor is Encorafenib (Compound A29) or a compound disclosed in PCT Publication No. WO 2011/025927. In one embodiment, a GITR antibody molecule is used in combination with Encorafenib (Compound A29), or a compound disclosed in PCT Publication No. WO 2011/025927, to treat a disorder such as a non-small cell lung cancer, a melanoma, or a colorectal cancer.

In one embodiment, the BRAF inhibitor or Encorafenib (Compound A29) is administered at a dose of about 200-300, 200-400, or 300-400 mg, e.g., per day. In one embodiment, the compound is administered at a dose of about 200, about 300 or about 400 mg.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination a CDK4/6 inhibitor, 7-cyclopentyl-N,N-dimethyl-2-((5-((1R,6S)-9-methyl-4-oxo-3,9-diazabicyclo[4.2.1]nonan-3-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound A30), or a compound disclosed in PCT publication No. WO 2011/101409 to treat a disorder, e.g., a disorder described herein. In one embodiment, the CDK4/6 inhibitor is 7-cyclopentyl-N,N-dimethyl-2-((5-((1R,6S)-9-methyl-4-oxo-3,9-diazabicyclo[4.2.1]nonan-3-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound A30) or a compound disclosed in PCT publication No. WO 2011/101409. In one embodiment, a GITR antibody molecule is used in combination with 7-cyclopentyl-N,N-dimethyl-2-((5-((1R,6S)-9-methyl-4-oxo-3,9-diazabicyclo[4.2.1]nonan-3-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound A30), or a compound disclosed in PCT publication No. WO 2011/101409, to treat a disorder such as a cancer, a mantle cell lymphoma, a liposarcoma, a non-small cell lung cancer, a melanoma, a squamous cell esophageal cancer, or a breast cancer.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a HER3 inhibitor, Compound A31, or a compound disclosed in PCT Publication No. WO 2012/022814, to treat a disorder, e.g., a disorder described herein. In one embodiment, the HER3 inhibitor is Compound A31 or a compound disclosed in PCT Publication WO 2012/022814. In one embodiment, a GITR antibody molecule is used in combination with Compound A31, or a compound disclosed in PCT Publication WO 2012/022814, to treat a disorder such as a gastric cancer, an esophageal cancer, a head and neck cancer, a squamous cell carcinoma, a stomach cancer, a breast cancer (e.g., metastatic breast cancer), or a digestive/gastrointestinal cancer.

In some embodiments, Compound A31 is a human monoclonal antibody molecule.

In one embodiment, the HER3 inhibitor or Compound A31 is administered at a dose of about 3, 10, 20, or 40 mg/kg, e.g., once weekly (QW). In one embodiment, the compound is administered at a dose of about 3-10, 10-20, or 20-40 mg/kg.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination an FGFR2 and/or FGFR4 inhibitor, Compound A32, or a compound disclosed in a publication PCT Publication No. WO 2014/160160 (e.g., an antibody molecule drug conjugate against an FGFR2 and/or FGFR4, e.g., mAb 12425), to treat a disorder, e.g., a disorder described herein. In one embodiment, the FGFR2 and/or FGFR4 inhibitor is Compound A32 or a compound disclosed in a publication PCT Publication No. WO 2014/160160. In one embodiment, a GITR antibody molecule is used in combination with Compound A32, or a compound as described in Table 6, to treat a disorder such as a cancer, a gastric cancer, a breast cancer, a rhabdomyosarcoma, a liver cancer, an adrenal cancer, a lung cancer, an esophageal cancer, a colon cancer, or an endometrial cancer.

In some embodiments, Compound A32 is an antibody molecule drug conjugate against an FGFR2 and/or FGFR4, e.g., mAb 12425.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination an M-CSF inhibitor, Compound A33, or a compound disclosed in PCT Publication No. WO 2004/045532 (e.g., an antibody molecule or Fab fragment against M-CSF), to treat a disorder, e.g., a disorder described herein.

In one embodiment, the M-CSF inhibitor is Compound A33 or a compound disclosed in PCT Publication No. WO 2004/045532. In one embodiment, a GITR antibody molecule is used in combination with Compound A33, or a compound as described in PCT Publication No. WO 2004/045532, to treat a disorder such as a cancer, a prostate cancer, a breast cancer, or pigmented villonodular synovitis (PVNS).

In embodiments, Compound A33 is a monoclonal antibody molecule against M-CSF or a fragment (e.g., Fab fragment) thereof. In embodiments, the M-CSF inhibitor or Compound A33 is administered at an average dose of about 10 mg/kg.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a MEK inhibitor, Binimetinib (Compound A34), or a compound disclosed in PCT Publication No. WO 2003/077914 to treat a disorder, e.g., a disorder described herein. In one embodiment, the MEK inhibitor is Binimetinib (Compound A34), or a compound disclosed in PCT Publication No. WO 2003/077914. In one embodiment, a GITR antibody molecule is used in combination with Binimetinib (Compound A34), or a compound disclosed in PCT Publication No. WO 2003/077914, to treat a disorder such as a non-small cell lung cancer, a multisystem genetic disorder, a melanoma, an ovarian cancer, a digestive/gastrointestinal cancer, a rheumatoid arthritis, or a colorectal cancer.

In one embodiment, the MEK inhibitor or Binimetinib (Compound A34) is administered at a dose of about 45 mg, e.g., twice daily.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination an inhibitor of one or more of c-KIT, histamine release, Flt3 (e.g., FLK2/STK1) or PKC, Midostaurin (Compound A35) or a compound disclosed in PCT Publication No. WO 2003/037347 to treat a disorder, e.g., a disorder described herein. In one embodiment, the inhibitor is Midostaurin (Compound A35) or compound disclosed in PCT Publication No. WO 2003/037347. In one embodiment, the inhibitor of one or more of c-KIT, histamine release, Flt3 (e.g., FLK2/STK1) or PKC is Midostaurin. In one embodiment, a GITR antibody molecule is used in combination with Midostaurin (Compound A35), or compound disclosed in PCT Publication No. WO 2003/037347, to treat a disorder such as a cancer, a colorectal cancer, a myeloid leukemia, myelodysplastic syndrome, an age-related mascular degeration, a diabetic complication, or a dermatologic disorder.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a TOR inhibitor (e.g., mTOR inhibitor), Everolimus (also known as AFINITOR; Compound A36) or a Compound disclosed in PCT Publication No. WO 2014/085318 to treat a disorder, e.g., a disorder described herein). In one embodiment, the TOR inhibitor is Everolimus (Compound A36) or a Compound disclosed in PCT Publication No. WO 2014/085318. In one embodiment, a GITR antibody molecule is used in combination with Everolimus (Compound A36) to treat a disorder such as an interstitial lung disease, a small cell lung cancer, a respiratory/thoracic cancer, a prostate cancer, a multiple myeloma, a sarcoma, an age-related macular degeneration, a bone cancer, tuberous sclerosis, a non-small cell lung cancer, an endocrine cancer, a lymphoma, a neurologic disorders, an astrocytoma, a cervical cancer, a neurologic cancer, a leukemia, an immune disorders, transplant rejection, a gastric cancer, a melanoma, epilepsy, a breast cancer, or a bladder cancer.

In one embodiment, the TOR inhibitor or Everolimusis (Compound A36) administered at a dose of about 2.5-20 mg/day. In one embodiment, the compound is administered at a dose of about 2.5, 5, 10, or 20 mg/day, e.g., about 2.5-5, 5-10, or 10-20 mg/day.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination an inhibitor of one or more of VEGFR-2, PDGFRbeta, KIT or Raf kinase C, 1-methyl-5-((2-(5-(trifluoromethyl)-1H-imidazol-2-yl)pyridin-4-yl)oxy)-N-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-2-amine (Compound A37) or a compound disclosed in PCT Publication No. WO 2007/030377 to treat a disorder, e.g., a disorder described herein. In one embodiment, the inhibitor of one or more of VEGFR-2, PDGFRbeta, KIT or Raf kinase C is 1-methyl-5-((2-(5-(trifluoromethyl)-1H-imidazol-2-yl)pyridin-4-yl)oxy)-N-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-2-amine (Compound A37) or a compound disclosed in PCT Publication No. WO 2007/030377. In one embodiment, a GITR antibody molecule is used in combination with 1-methyl-5-((2-(5-(trifluoromethyl)-1H-imidazol-2-yl)pyridin-4-yl)oxy)-N-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-2-amine (Compound A37), or a compound disclosed in PCT Publication No. WO 2007/030377, to treat a disorder such as a cancer, a melanoma, or a solid tumor.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination a somatostatin agonist and/or growth hormone release inhibitor, Pasireotide diaspartate (also known as SIGNIFOR; Compound A38) or a compound disclosed in PCT Publication No. WO2002/010192 or U.S. Pat. No. 7,473,761 to treat a disorder, e.g., a disorder described herein. In one embodiment, the somatostatin agonist and/or growth hormone release inhibitor is Pasireotide diaspartate (Compound A38) or a compound disclosed in PCT Publication No. WO2002/010192 or U.S. Pat. No. 7,473,761. In one embodiment, a GITR antibody molecule is used in combination with Pasireotide diaspartate (Compound A38), or a compound disclosed in PCT Publication No. WO2002/010192 or U.S. Pat. No. 7,473,761, to treat a disorder such as a prostate cancer, an endocrine cancer, a nurologic cancer, a skin cancer (e.g., a melanoma), a pancreatic cancer, a liver cancer, Cushing's syndrome, a gastrointestinal disorder, acromegaly, a liver and biliary tract disorder, or liver cirrhosis.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination a signal transduction modulator and/or angiogenesis inhibitor, Dovitinib (Compound A39) or a compound disclosed in PCT Publication No. WO 2009/115562 to treat a disorder, e.g., a disorder described herein. In one embodiment, the signal transduction modulator and/or angiogenesis inhibitor is Dovitinib (Compound A39) or a compound disclosed in PCT Publication No. WO 2009/115562. In one embodiment, a GITR antibody molecule is used in combination with Dovitinib (Compound A39), or a compound disclosed in PCT Publication No. WO 2009/115562, to treat a disorder such as a cancer, a respiratory/thoracic cancer, a multiple myeloma, a prostate cancer, a non-small cell lung cancer, an endocrine cancer, or a neurological genetic disorder.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an EGFR inhibitor, (R,E)-N-(7-chloro-1-(1-(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (Compound A40) or a compound disclosed in PCT Publication No. WO 2013/184757 to treat a disorder, e.g., a disorder described herein. In one embodiment, the EGFR inhibitor is (R,E)-N-(7-chloro-1-(1-(4-(dimethylamino) but-2-enoyl)azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (Compound A40) or a compound disclosed in PCT Publication No. WO 2013/184757. In one embodiment, a GITR antibody molecule is used in combination with (R,E)-N-(7-chloro-1-(1-(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (Compound A40), or a compound disclosed in PCT Publication No. WO 2013/184757, to treat a disorder such as a cancer, e.g., a solid tumor.

In one embodiment, the EGFR inhibitor or (R,E)-N-(7-chloro-1-(1-(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (Compound A40) is administered at a dose of 150-250 mg, e.g., per day. In one embodiment, the compound is administered at a dose of about 150, 200, or 250 mg, or about 150-200 or 200-250 mg.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination an ALK inhibitor, N⁶-(2-isopropoxy-5-methyl-4-(1-methylpiperidin-4-yl)phenyl)-N⁴-(2-(isopropylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (Compound A42) or a compound disclosed in PCT Publication No. WO 2008/073687 to treat a disorder, e.g., a disorder described herein. In one embodiment, the ALK inhibitor is N⁶-(2-isopropoxy-5-methyl-4-(1-methylpiperidin-4-yl)phenyl)-N⁴-(2-(isopropylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (Compound A42) or a compound disclosed in PCT Publication No. WO 2008/073687. In one embodiment, a GITR antibody molecule is used in combination with N⁶-(2-isopropoxy-5-methyl-4-(1-methylpiperidin-4-yl)phenyl)-N⁴-(2-(isopropylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (Compound A42), or a compound disclosed in PCT Publication No. WO 2008/073687, to treat a disorder such as a cancer, an anaplastic large-cell lymphoma (ALCL), a non-small cell lung carcinoma (NSCLC), or a neuroblastoma.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination an IGF-1R inhibitor, 3-(4-(4-((5-chloro-4-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)amino)-5-fluoro-2-methylphenyl)piperidin-1-yl)thietane 1,1-dioxide (Compound A43), 5-chloro-N²-(2-fluoro-5-methyl-4-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)phenyl)-N⁴-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (Compound A44), or 5-chloro-N2-(4-(1-ethylpiperidin-4-yl)-2-fluoro-5-methylphenyl)-N⁴-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (Compound A45) or a compound disclosed in PCT Publication No. WO 2010/002655 to treat a disorder, e.g., a disorder described. In one embodiment, the IGF-1R inhibitor is 3-(4-(4-((5-chloro-4-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)amino)-5-fluoro-2-methylphenyl)piperidin-1-yl)thietane 1,1-dioxide (Compound A43), 5-chloro-N²-(2-fluoro-5-methyl-4-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)phenyl)-N⁴-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (Compound A44), 5-chloro-N2-(4-(1-ethylpiperidin-4-yl)-2-fluoro-5-methylphenyl)-N⁴-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (Compound A45), or a compound disclosed in PCT Publication No. WO 2010/002655. In one embodiment, a GITR antibody molecule is used in combination with 3-(4-(4-((5-chloro-4-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)amino)-5-fluoro-2-methylphenyl)piperidin-1-yl)thietane 1,1-dioxide (Compound A43), 5-chloro-N²-(2-fluoro-5-methyl-4-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)phenyl)-N⁴-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (Compound A44), 5-chloro-N2-(4-(1-ethylpiperidin-4-yl)-2-fluoro-5-methylphenyl)-N⁴-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (Compound A45), or a compound disclosed in PCT Publication No. WO 2010/002655, to treat a disorder such as a cancer or a sarcoma.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination a P-Glycoprotein 1 inhibitor, Valspodar (also known as AMDRAY; Compound A46) or a compound disclosed in EP 296122 to treat a disorder, e.g., a disorder described herein. In one embodiment, the P-Glycoprotein 1 inhibitor is Valspodar (Compound A46) or a compound disclosed in EP 296122. In one embodiment, a GITR antibody molecule is used in combination with Valspodar (Compound A46), or a compound disclosed in EP 296122, to treat a disorder such as a cancer or a drug-resistant tumor.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination one or more of a VEGFR inhibitor, Vatalanib succinate (Compound A47) or a compound disclosed in EP 296122 to treat a disorder, e.g., a disorder described herein. In one embodiment, the VEGFR inhibitor is Vatalanib succinate (Compound A47) or a compound disclosed in EP 296122. In one embodiment, a GITR antibody molecule is used in combination with Vatalanib succinate (Compound A47), or a compound disclosed in EP 296122, to treat cancer.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an IDH inhibitor or a compound disclosed in WO2014/141104 to treat a disorder, e.g., a disorder described herein. In one embodiment, the IDH inhibitor is a compound disclosed in PCT Publication No.

WO2014/141104. In one embodiment, a GITR antibody molecule is used in combination with a compound disclosed in WO2014/141104 to treat a disorder such as a cancer.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a BCL-ABL inhibitor or a compound disclosed in PCT Publication No. WO2013/171639, WO2013/171640, WO2013/171641, or WO2013/171642 to treat a disorder, e.g., a disorder described herein. In one embodiment, the BCL-ABL inhibitor is a compound disclosed in PCT Publication No. WO2013/171639, WO2013/171640, WO2013/171641, or WO2013/171642. In one embodiment, a GITR antibody molecule is used in combination with a compound disclosed in PCT Publication No. WO2013/171639, WO2013/171640, WO2013/171641, or WO2013/171642 to treat a disorder such as a cancer.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with a c-RAF inhibitor or a compound disclosed in PCT Publication No. WO2014/151616 to treat a disorder, e.g., a disorder described herein. In one embodiment, the c-RAF inhibitor is Compound A50 or a compound disclosed in PCT Publication No. WO2014/151616. In one embodiment, a GITR antibody molecule is used in combination with a compound disclosed in PCT Publication No. WO2014/151616 to treat a disorder such as a cancer.

In another embodiment, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is used in combination with an ERK1/2 ATP competitive inhibitor or a compound disclosed in International Patent Application No. PCT/US2014/062913 to treat a disorder, e.g., a disorder described herein. In one embodiment, the ERK1/2 ATP competitive inhibitor is a compound disclosed in PCT Publication No. WO2015/066188. In one embodiment, a GITR antibody molecule is used in combination with Compound A51 or a compound disclosed in PCT Publication No. WO2015/066188 to treat a disorder such as a cancer.

In some embodiments, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is administerd in combination with one or more agents selected from, Compound A8, Compound A17, Compound A23, Compound A24, Compound A27, Compound A29, and Compound A33.

In some embodiments, the combination, e.g., a combination comprising an anti-GITR antibody molecule as described herein, is administered in combination with an anti-cancer agent having a known activity in an immune cell assay, e.g., in one or more of a huMLR assay, a T cell proliferation assay, and a B-cell proliferation assay. Exemplary assays are described below. Based on the assay, an IC50 for can be calculated for each test agent. In embodiments, the anti-cancer agent has an IC50 of, e.g., 0-1 μM, 1-4 μM, or greater than 4 μM, e.g., 4-10 μM or 4-20 μM. In embodiments, the second therapeutic agent is chosen from one or more of: Compound A9, Compound A16, Compound A17, Compound A21, Compound A22, Compound A25, Compound A28, Compound A48, and Compound 49.

In some embodiments, the Compound A28 (or a compound related to Compound A28) is administered at a dose of approximately 5-10 or 10-30 mg. In some embodiments, the Compound A22 (or compound related to Compound A22) is administered at a dose of about 200 mg. In some embodiments, the Compound A17 (or compound related to Compound A17) is administered at a dose of approximately 400-600 mg. In some embodiments, the Compound A16 (or compound related to Compound A16) is administered at a dose of approximately 400-600 mg PO qDay. In some embodiments, the Compound A29 (or compound related to Compound A29) is administered at a dose of approximately 200-400 or 300-400 mg. In some embodiments, the Compound A24 (or compound related to Compound A24) is administered at a dose of approximately 200-600 mg. In some embodiments, the Compound A23 (ceritinib) (or compound related to ceritinib) is administered at a dose of approximately 750 mg once daily. In some embodiments, the Compound A8 (or compound related to Compound A8) is administered at a dose of approximately 200-400 or 300-400 mg. In some embodiments, the Compound A5 (or compound related to Compound A5) is administered at a dose of approximately 100-125 mg. In some embodiments, the Compound A6 (or compound related to Compound A6) is administered at a dose of about 100 mg. In some embodiments, the Compound A1 (or compound related to Compound A1) is administered at a dose of approximately 200-300 or 200-600 mg. In some embodiments, the Compound A40 (or compound related to Compound A40) is administered at a dose of approximately 150-250 mg. In embodiments, the Compound A10 (or compound related to Compound A10) is administered at a dose of approximately 400 to 700 mg, e.g., administered three times weekly, 2 weeks on and one week off. In embodiments, the BCR-ABL inhibitor is administered at a dose of approximately 20 mg bid-80 mg bid.

TABLE 6 Selected therapeutic agents that can be administered in combination with the anti-GITR antibody molecules provided herein, e.g., as a single agent or in combination with other immunomodulators described herein (e.g., one or more of: an activator of a costimulatory molecule and/or an inhibitor of an immune checkpoint molecule). Each publication listed in this Table is herein incorporated by reference in its entirety, including all structural formulae therein. Patents/Patent Compound Generic Name Application Designation Tradename Compound Structure Publications A1 Sotrastaurin

EP 1682103 US 2007/142401 WO 2005/039549 A2 Nilotinib HCl monohydrate TASIGNA ®

WO 2004/005281 U.S. Pat. No. 7,169,791 A3

WO 2010/060937 WO 2004/072051 EP 1611112 U.S. Pat. No. 8,450,310 A4 Dactolisib

WO 2006/122806 A5

U.S. Pat. No. 8,552,002 A6 Buparlisib

WO 2007/084786 A7

WO 2009/141386 US 2010/0105667 A8

WO 2010/029082 A9 CYP17 inhibitor WO 2010/149755 U.S. Pat. No. 8,263,635 B2 EP 2445903 B1 A10

WO 2011/076786 A11 Deferasirox EXJADE ®

WO 1997/049395 A12 Letrozole FEMARA ®

U.S. Pat. No. 4,978,672 A13

WO 2013/124826 US 2013/0225574 A14

WO 2013/111105 A15

WO 2005/073224 A16 Imatinib mesylate GLEEVEC ®

WO 1999/003854 A17

EP 2099447 U.S. Pat. No. 7,767,675 U.S. Pat. No. 8,420,645 A18 Ruxolitinib Phosphate JAKAFI ®

WO 2007/070514 EP 2474545 U.S. Pat. No. 7,598,257 WO 2014/018632 A19 Panobinostat

WO 2014/072493 WO 2002/022577 EP 1870399 A20 Osilodrostat

WO 2007/024945 A21

WO 2008/016893 EP 2051990 U.S. Pat. No. 8,546,336 A22 Sonidegib phosphate

WO 2007/131201 EP 2021328 U.S. Pat. No. 8,178,563 A23 ceritinib ZYKADIA ®

WO 2008/073687 U.S. Pat. No. 8,039,479 A24

U.S. Pat. No. 8,415,355 8,685,980 A25

WO 2010/007120 A26 Human monoclonal antibody to PRLR U.S. Pat. No. 7,867,493 A27

WO 2010/026124 EP 2344474 US 2010/0056576 WO2008/106692 A28

WO 2010/101849 A29 Encorafenib

WO 2011/025927 A30

WO 2011/101409 A31 Human monoclonal antibody to HER3 WO 2012/022814 EP 2606070 U.S. Pat. No. 8,735,551 A32 Antibody Drug Conjugate (ADC) WO 2014/160160 Ab: 12425 (see Table 1, paragraph [00191]) Linker: SMCC (see paragraph [00117] Pay load: DM1 (see paragraph [00111] See also Claim 29 A33 Monoclonal antibody or Fab to M-CSF WO 2004/045532 A34 Binimetinib

WO 2003/077914 A35 midostaurin

WO 2003/037347 EP 1441737 US 2012/252785 A36 Everolimus AFINITOR ®

WO 2014/085318 A37

WO 2007/030377 U.S. Pat. No. 7,482.367 A38 Pasireotide diaspartate SIGNIFOR ®

WO2002/010192 U.S. Pat. No. 7,473.761 A39 Divitinib

WO 2009/115562 U.S. Pat. No. 8,563.556 A40

WO 2013/184757 A41

WO 2006/122806 A42

WO 2008/073687 U.S. Pat. No. 8,372.858 A43

WO 2010/002655 8,519.129 A44

WO 2010/002655 U.S. Pat. No. 8,519,129 A45

WO 2010/002655 A46 Valspodar AMDRAY ™

EP 296122 A47 Vatalanib succinate

WO 98/35958 A48 IDH inhibitor WO2014/141104 A49 BCR-ABL inhibitor WO2013/171639 WO2013/171640 WO2013/171641 WO2013/171642 A50 cRAF inhibitor WO2014/151616 A51 ERK1/2 ATP competitive inhibitor WO2015066188 Table 6 provides from left to right the following: the Compound Designation of the second therapeutic agent, the Compound structure, and Patent publication(s) disclosing the Compound.

Combinations of Antigen-Presentation Combinations, Effector Cell Combinations and Anti-Tumor Immunosuppression Agents

Provided herein, at least in part, are methods and compositions comprising a combination of two, three or more therapeutic agents chosen from one, two, or all of the following categories (i)-(iii): (i) an agent that enhances antigen presentation (e.g., tumor antigen presentation); (ii) an agent that enhances an effector cell response (e.g., B cell and/or T cell activation and/or mobilization); or (iii) an agent that decreases tumor immunosuppression, wherein at least one therapeutic agent is a GITR antibody, antibody fragment or antigen binding agent provided herein.

Without wishing to be bound by theory, it is believed that therapeutic approaches that enhance anti-tumor immunity work more effectively when the immune response is optimized by targeting multiple components at one or more stages of an immune response, e.g., an anti-tumor immune response. For example, approaches that enhance antigen presentation, e.g., by activation and/or maturation of dendritic cells, combined with approaches that enhance cellular and humoral immune responses (e.g., by stimulating, e.g., disinhibiting, phagocytes and/or tumor infiltrating lymphocytes (e.g., NK cells and T cells)), while blocking tumor immunosuppressive signaling (e.g., by increasing macrophage polarization, increasing T_(reg) depletion and/or decreasing myeloid-derived suppressive cells (MDSCs)) can result in a more effective and/or prolonged therapeutic response. Accordingly, disclosed herein are combination therapies that optimize one, two, or all of: (i) antigen presentation, e.g., increasing antigen presentation (e.g., by enhancing one or more of dendritic cell activity or maturation, antigen uptake, or antigen processing); (ii) effector cell response, e.g., increasing effector cell response (e.g., enhancing B cell and/or T cell activation and/or mobilization, e.g., in the lymph node); or (iii) tumor immunosuppression, e.g., decreasing tumor immunosuppression (e.g., increasing T cell infiltration and tumor cell killing).

The combinations described herein can provide a superior beneficial effect, e.g., in the treatment of a disorder, such as an enhanced anti-cancer effect, reduced toxicity and/or reduced side effects, compared to monotherapy administration of the therapeutic agents in the combination. For example, one or more of the therapeutic agents in the combination can be administered at a lower dosage, or for a shorter period of administration, than would be required to achieve the same therapeutic effect compared to the monotherapy administration. Thus, compositions and methods for treating cancer and other immune disorders using the aforesaid combination therapies are disclosed.

Accordingly, in one aspect, the invention features a method of treating (e.g., inhibiting, reducing, ameliorating, or preventing) a disorder, e.g., a hyperproliferative condition or disorder (e.g., a cancer) in a subject. The method includes administering to the subject a combination of two, three or more therapeutic agents chosen from one, two or all of the following categories (i)-(iii): (i) an agent that enhances antigen (e.g., tumor antigen) presentation; (ii) an agent that enhances an effector cell response (e.g., B cell and/or T cell activation and/or mobilization); or (iii) an agent that decreases tumor immunosuppression, thereby treating the disorder, e.g., the hyperproliferative condition or disorder (e.g., the cancer). The combinations include a-GITR modulator (e.g., an anti-GITR antibody molecule as described herein). The cancer treated can be, e.g., a cancer described herein, such as lung cancer (squamous), lung cancer (adenocarcinoma), head and neck cancer, cervical cancer (squamous), stomach cancer, thyroid cancer, melanoma, nasopharyngeal cancer, or breast cancer.

Antigen Presentation Combinations

In certain embodiments, the combination includes one, two, three, four or more therapeutic agents that enhance antigen (e.g., tumor antigen) presentation (referred to herein as an “antigen-presentation combination”) in addition to a GITR modulator (e.g., an anti-GITR antibody molecule as described herein). In certain embodiments, the antigen presentation combination includes one or more of: an agent that enhances antigen presentation (e.g., a vaccine, e.g., a cell- or antigen-based vaccine); an agent that enhances lysis of tumor cells (e.g., an oncolytic virus); an agent that stimulates (e.g., disinhibits) a phagocyte, e.g., a Type I interferon (IFN) activator (e.g., a TLR agonist, a RIG-I-like receptor agonist (RLRs)), and/or an agent that activates and/or recruits a dendritic cell or a macrophage (e.g., a macrophage I), e.g., a bi- or tri-specific cell engager.

In some embodiments, the antigen-presentation combination includes one, two, three, four, five or more therapeutic agents chosen from: (i) an agonist of Stimulator of Interferon Genes (a STING agonist), (ii) an agonist of a Toll-like receptor (TLR) (e.g., an agonist of TLR-3, -4, -5, -7, -8, or -9), (iii) a TIM-3 modulator (e.g., an anti-TIM-3 antibody molecule), (iv) a vascular endothelial growth factor receptor (VEGFR) inhibitor, (v) a c-Met inhibitor, (vi) a TGFb inhibitor (e.g., an anti-TGFb antibody), (vii) an IDO/TDO inhibitor, (viii) an A2AR antagonist, (ix) an oncolytic virus, (x) a vaccine (e.g., a scaffold vaccine), or (xi) a bi- or tri-specific cell engager. Any combination of the aforesaid agents (i)-(xi) can be used in the antigen-presentation combination. In one exemplary embodiment, the antigen-presentation combination includes a STING agonist. In another exemplary embodiment, the antigen-presentation combination includes a TLR agonist (e.g., a TLR7 agonist). In another exemplary embodiment, the antigen-presentation combination includes a STING agonist and a TLR agonist (e.g., a TLR7 agonist). In some embodiments, the antigen presentation combination is chosen from a STING agonist, a TLR agonist, an A2AR antagonist, or an oncolytic virus or a combination thereof, and optionally, one or more of (iii)-(vii) or (x)-(xi). In some embodiments, the antigen presentation combination is chosen from a STING agonist or a TLR agonist, or a combination of both, and optionally, one or more of (iii)-(xi). In another embodiment, the antigen-presentation combination includes a STING agonist, a TLR agonist (e.g., a TLR7 agonist) and a TIM-3 modulator (e.g., an anti-TIM-3 inhibitor). In another embodiment, the antigen-presentation combination includes a STING agonist, a TLR agonist (e.g., a TLR7 agonist) and a VEGFR inhibitor. In another embodiment, the antigen-presentation combination includes a STING agonist, a TLR agonist (e.g., a TLR7 agonist) and a c-MET inhibitor. In yet other embodiments, the antigen-presenting combination includes an oncolytic virus. In other embodiments, the antigen-presenting combination includes an oncolytic virus and a cytokine, e.g., an oncolytic virus expressing one or more of GM-CSF, or a CSF (e.g., CSF1, or CSF2). In some embodiments, the antigen-presenting combination includes a bi- or tri-specific cell engager, e.g., a bi- or tri-specific antibody molecule to CD47 and CD19, with or without an Fc domain. In some embodiments, the antigen-presenting combination includes a TGFb inhibitor (e.g., an anti-TGFb antibody). In other embodiments, the antigen-presenting combination includes an IDO/TDO inhibitor. In yet other embodiments, the antigen-presenting combination includes an A2AR antagonist. In yet other embodiments, the antigen-presenting combination includes a vaccine (e.g., IL-2 in combination with MUC1, or a dendritic cell based vaccine (e.g., Provenge®)). In yet other embodiments, the antigen-presenting combination includes a vaccine and a TLR agonist (e.g., a TLR agonist as described herein). In certain embodiment, the antigen-presentation combination includes a vaccine and a STING agonist. In certain embodiment, the antigen-presentation combination includes a vaccine, a STING agonist and a TLR agonist.

Effector Cell Combinations

In certain embodiments, the combination includes one, two, three, four, five or more therapeutic agents that enhance an effector cell response (referred to herein as an “effector cell combination”) in addition to a GITR modulator (e.g., an anti-GITR antibody molecule as described herein). In some embodiments, the effector cell combination includes a lymphocyte activator, e.g., an NK cell activator and/or a T cell activator. In some embodiments, the effector cell combination activates (e.g., disinhibits) a tumor infiltrating lymphocyte (TIL), e.g., an NK cell or a T cell. In some embodiments, the effector cell combination includes an NK cell modulator chosen from a modulator (e.g., an antibody molecule) of an NK receptor (e.g., a modulator of one or more of NKG2A, KIR3DL, NKp46, MICA or CEACAM1); an interleukin or an interleukin variant (e.g., IL-2, IL-15, IL-21, IL-13R or IL-12 cytokine or variant thereof, or a combination thereof); a bi- or tri-specific cell engager (e.g., a bispecific antibody molecule of NKG2A and CD138, or a bispecific antibody molecule of CD3 and TCR); an NK cell therapy; or a vaccine that includes NK cells and an antigen/immune stimulant. In some embodiments, the effector cell combination includes an immunomodulator (e.g., one or more of: an activator of a costimulatory molecule or an inhibitor of an immune checkpoint molecule as described herein). In some embodiments, the effector cell combination includes a T cell modulator chosen from an inhibitor of a checkpoint inhibitor (e.g., an inhibitor of one or more of: PD-1, PD-L1, TIM-3, LAG-3, VISTA, DKG-α, B7-H3, B7-H4, TIGIT, CTLA4, BTLA, CD160, TIMI, IDO, LAIR1, IL-12, or a combination thereof, e.g., an inhibitor of PD-1 and TIM-3, or an inhibitor of PD-1 and LAG-3). In one embodiment, the inhibitor of the checkpoint inhibitor is an antibody molecule (e.g., a mono- or bispecific antibody or fragment thereof as described herein). For example, the inhibitor of the checkpoint inhibitor is an antibody molecule against PD-1, PD-L1, TIM-3, LAG-3, VISTA, B7-H4, CTLA4 or TIGIT, or any combination thereof (e.g. a combination as described herein). In some embodiments, the effector cell combination includes a T cell modulator chosen from an agonist or an activator of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is chosen from an agonist (e.g., an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion) of GITR, OX40, ICOS, SLAM (e.g., SLAMF7), HVEM, LIGHT, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, CD7, NKG2C, NKp80, CD160, B7-H3, or CD83 ligand. In other embodiments, the effector cell combination includes a bispecific T cell engager (e.g., a bispecific antibody molecule that binds to CD3 and a tumor antigen (e.g., EGFR, PSCA, PSMA, EpCAM, HER2 among others).

In some embodiments, the effector cell combination includes one, two, three, four, five or more therapeutic agents chosen from: (i) a GITR modulator (e.g., a GITR agonist (e.g., an anti-GITR antibody molecule as described herein)), (ii) a PD-1 inhibitor, (iii) a PD-L1 inhibitor, (iv) an inhibitor of IAP (Inhibitor of Apoptosis Protein), (v) an inhibitor of EGFR (Epidermal Growth Factor Receptor), (vi) an inhibitor of target of rapamycin (mTOR), (vii) IL-15 or a variant thereof, (viii) a CTLA-4 inhibitor, (ix) a bispecific T cell engager (e.g., a bispecific antibody molecule that binds to CD3 and a tumor antigen (e.g., EGFR, PSCA, PSMA, EpCAM, HER2 among others), (x) a CD40 agonist (e.g., an anti-CD40 antibody molecule), (xi) an OX40 agonist (e.g., an anti-OX40 antibody molecule), or (xii) a CD27 agonist (e.g., an anti-CD27 antibody molecule). Any combination of the aforesaid agents can be used in the effector cell combination. In one exemplary embodiment, the effector cell combination includes a GITR agonist (e.g., an anti-GITR antibody molecule as described herein). In another embodiment, the effector cell combination includes a PD-1 inhibitor. In another embodiment, the effector cell combination includes a PD-L1 inhibitor. In other embodiments, the effector cell combination includes a GITR agonist (e.g., an anti-GITR antibody molecule as described herein) and a PD-1 inhibitor. In other embodiments, the effector cell combination includes a GITR agonist (e.g., an anti-GITR antibody molecule as described herein) and a PD-L1 inhibitor. In other embodiments, the effector cell combination includes a GITR agonist (e.g., an anti-GITR antibody molecule as described herein), a PD-1 inhibitor, and a PD-L1 inhibitor. In other embodiments, the effector cell combination includes a PD-1 inhibitor, and a PD-L1 inhibitor. In one embodiment, the effector cell combination includes a GITR agonist (e.g., an anti-GITR antibody molecule as described herein) and an inhibitor of IAP. In another embodiment, the effector cell combination includes a GITR agonist (e.g., an anti-GITR antibody molecule as described herein) and an inhibitor of an EGFR inhibitor. In yet another embodiment, the effector cell combination includes a GITR agonist (e.g., an anti-GITR antibody molecule as described herein) and an inhibitor of an mTOR inhibitor. In one embodiment, the effector cell combination includes IL-15 or a variant thereof. In one embodiment, the effector cell combination includes a CTLA-4 inhibitor. In one embodiment, the effector cell combination includes a bispecific T cell engager (e.g., a bispecific antibody molecule that binds to CD3 and a tumor antigen (e.g., EGFR, PSCA, PSMA, EpCAM, HER2 among others). In one embodiment, the effector cell combination includes a CD40 agonist (e.g., an anti-CD40 antibody molecule). In one embodiment, the effector cell combination includes an OX40 agonist (e.g., an anti-OX40 antibody molecule). In one embodiment, the effector cell combination includes a CD27 agonist (e.g., an anti-CD27 antibody molecule).

Anti-Tumor Immunosuppression Combinations

In certain embodiments, the combination includes one, two, three, four, five or more therapeutic agents that decrease tumor immunosuppression (referred to herein as an “anti-tumor immunosuppression combination”) in addition to a GITR modulator (e.g., an anti-GITR antibody molecule as described herein). In some embodiments, the combination modulates the activity or level of one or more of T_(reg), macrophage 2 or MDSCs. In some embodiments, the combination increases one or more of M2 polarization, T_(reg) depletion, or T cell recruitment. In some embodiments, the anti-tumor immunosuppression combination includes one, two, three, four, five or more therapeutic agents chosen from: (i) an immunomodulator (e.g., one or more of: an activator of a costimulatory molecule (e.g., a GITR agonist), or an inhibitor of an immune checkpoint molecule (e.g., one or more of PD-1, PD-L1, LAG-3, TIM-3 or CTLA-4), as described herein), (ii) a CSF-1/1R inhibitor (e.g., an inhibitor of macrophage colony-stimulating factor (M-CSF)), (iii) an IL-17 inhibitor, (iv) an IL-1β inhibitor, (v) a CXCR2 inhibitor, (vi) an inhibitor of a phosphoinositide 3-kinase (PI3K, e.g., PI3Kγ or PI3Kδ), (vii) a BAFF-R inhibitor, (viii) a MALT-1/BTK inhibitor, (ix) a JAK inhibitor, (x) a CRTH2 inhibitor, (xi) a VEGFR inhibitor, (xiii) an IL-15 or a variant thereof, (xiv) a CTLA-4 inhibitor, (xv) an IDO/TDO inhibitor, (xvi) an A2AR antagonist, (xvii) a TGFb inhibitor, or (xviii) a PFKFB3 inhibitor. In certain embodiments, the immunomodulator is an inhibitor of an immune checkpoint molecule (e.g., an inhibitor of PD-1, PD-L1, LAG-3, TIM-3, CEACAM (e.g., CEACAM-1, -3 and/or -5), or CTLA-4, or any combination thereof). Any combination of the aforesaid agents can be used in the tumor immunosuppression combination. In one exemplary embodiment, the anti-tumor immunosuppression combination includes one, two, three, four, five or more therapeutic agents chosen from a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a TIM-3 modulator (e.g., an anti-TIM-3 inhibitor), a GITR agonist (e.g., an anti-GITR antibody molecule as described herein), a CSF-1/1R inhibitor (e.g., an M-CSF inhibitor), an IL-17 inhibitor, an IL-1β inhibitor, or a CXCR2 inhibitor. In one embodiment, the anti-tumor immunosuppression combination includes one, two, or all of a GITR agonist (e.g., an anti-GITR antibody molecule as described herein), CSF-1/1R inhibitor (e.g., an M-CSF inhibitor), an IL-17 inhibitor, an IL-1β inhibitor. In one embodiment, the anti-tumor immunosuppression combination includes a a GITR agonist (e.g., an anti-GITR antibody molecule as described herein), an IL-17 inhibitor, a CXCR2 inhibitor, a CRTH2 inhibitor, an A2AR antagonist, or a PFKFB3 inhibitor, or a combination thereof.

In some embodiments, the combination includes one or more therapeutic agents of the antigen-presentation combination. In other embodiments, the combination includes one or more therapeutic agents of the effector cell combination. In yet other embodiments, the combination includes one or more therapeutic agents of the anti-tumor immunosuppression combination. In other embodiments, the combination includes one or more therapeutic agents of the antigen-presentation combination and one or more therapeutic agents of the effector cell combination. In other embodiments, the one or more therapeutic agents of the antigen-presentation combination and one or more therapeutic agents of the anti-tumor immunosuppression combination. In other embodiments, the combination includes one or more therapeutic agents of the antigen-presentation combination, one or more therapeutic agents of the effector cell combination and one or more therapeutic agents of the anti-tumor immunosuppression combination. In other embodiments, the combination includes one or more therapeutic agents of the antigen-presentation combination, one or more therapeutic agents of the effector cell combination and one or more therapeutic agents of the anti-tumor immunosuppression combination.

Provided herein are methods and compositions that include a combination of one or more of: (i) an agent that enhances antigen (e.g., tumor antigen) presentation; (ii) an agent that enhances an effector cell response (e.g., B cell and/or T cell activation and/or mobilization); or (iii) an agent that decreases tumor immunosuppression, thereby treating the disorder, e.g., the hyperproliferative condition or disorder (e.g., the cancer). Provided combinations include a GITR modulator (e.g., an anti-GITR antibody molecule as described herein). Exemplary combinations of therapeutic agents from two or more of Antigen-Presentation Combinations category (A), Effector Cell Combinations category (B), and Anti-tumor Immunosuppression Combinations category (C) are provided in Table 7:

TABLE 7 Listing of Therapeutic Agents in Categories (A)-(C) A = Antigen- C = Anti-tumor Presentation B = Effector Cell Immunosuppression 1 STING agonist GITR agonist PD-1 inhibitor 2 TLR agonist PD-1 inhibitor PD-L1 inhibitor 3 TIM-3 modulator PD-L1 inhibitor LAG-3 inhibitor 4 VEGFR inhibitor IAP inhibitor TIM-3 inhibitor 5 c-MET inhibitor EGFR inhibitor GITR inhibitor 6 TGFb inhibitor mTOR inhibitor CSF-1/1R inhibitor 7 IDO/TDO inhibitor IL-15 agonist IL-17 inhibitor 8 A2AR antagonist CTLA-4 inhibitor IL-1β inhibitor 9 Oncolytic viruses Bispecific T-cell CXCR2 inhibitor engagers 10 Scaffold vaccines CD40 agonist PI3K-γ, -δ inhibitor 11 Bispecific T-cell OX40 agonist BAFF-R inhibitor engagers 12 CD27 agonist MALT-1/BTK inhibitor 13 JAK inhibitor 14 CRTH2 inhibitor 15 VEGFR inhibitor 16 IL-15 agonist 17 Anti-TGFb inhibitor 18 IDO/TDO inhibitor 19 A2AR antagonist 20 CTLA-4 inhibitor 21 PFKFB3 inhibitor

In some embodiments, the combinations of the present invention include one or more of the following (designations indicate combinations of agents provided in the matrix in Table 7): A1B1, A1B2, A1B3, A1B4, A1B5, A1B6, A1B7, A1B8, A1B9, A1B10, A1B11, A1B12, A2B1, A2B2, A2B3, A2B4, A2B5, A2B6, A2B7, A2B8, A2B9, A2B10, A2B11, A2B12, A3B1, A3B2, A3B3, A3B4, A3B5, A3B6, A3B7, A3B8, A3B9, A3B10, A3B11, A3B12, A4B1, A4B2, A4B3, A4B4, A4B5, A4B6, A4B7, A4B8, A4B9, A4B10, A4B11, A4B12, A5B1, A5B2, A5B3, A5B4, A5B5, A5B6, A5B7, A5B8, A5B9, A5B10, A5B11, A5B12, A6B1, A6B2, A6B3, A6B4, A6B5, A6B6, A6B7, A6B8, A6B9, A6B10, A6B11, A6B12, A7B1, A7B2, A7B3, A7B4, A7B5, A7B6, A7B7, A7B8, A7B9, A7B10, A7B11, A7B12, A8B1, A8B2, A8B3, A8B4, A8B5, A8B6, A8B7, A8B8, A8B9, A8B10, A8B11, A8B12, A9B1, A9B2, A9B3, A9B4, A9B5, A9B6, A9B7, A9B8, A9B9, A9B10, A9B11, A9B12, A10B1, A10B2, A10B3, A10B4, A10B5, A10B6, A10B7, A10B8, A10B9, A10B10, A10B11, A10B12, A11B1, A11B2, A11B3, A11B4, A11B5, A11B6, A11B7, A11B8, A11B9, A11B10, A11B11, A11B12, A1C1, A1C2, A1C3, A1C4, A1C5, A1C6, A1C7, A1C8, A1C9, A1C10, A1C11, A1C12, A1C13, A1C14, A1C15, A1C16, A1C17, A1C18, A1C19, A1C20, A1C21, A2C1, A2C2, A2C3, A2C4, A2C5, A2C6, A2C7, A2C8, A2C9, A2C10, A2C11, A2C12, A2C13, A2C14, A2C15, A2C16, A2C17, A2C18, A2C19, A2C20, A2C21, A3C1, A3C2, A3C3, A3C4, A3C5, A3C6, A3C7, A3C8, A3C9, A3C10, A3C11, A3C12, A3C13, A3C14, A3C15, A3C16, A3C17, A3C18, A3C19, A3C20, A3C21, A4C1, A4C2, A4C3, A4C4, A4C5, A4C6, A4C7, A4C8, A4C9, A4C10, A4C11, A4C12, A4C13, A4C14, A4C15, A4C16, A4C17, A4C18, A4C19, A4C20, A4C21, A5C1, A5C2, A5C3, A5C4, A5C5, A5C6, A5C7, A5C8, A5C9, A5C10, A5C11, A5C12, A5C13, A5C14, A5C15, A5C16, A5C17, A5C18, A5C19, A5C20, A5C21, A6C1, A6C2, A6C3, A6C4, A6C5, A6C6, A6C7, A6C8, A6C9, A6C10, A6C11, A6C12, A6C13, A6C14, A6C15, A6C16, A6C17, A6C18, A6C19, A6C20, A6C21, A7C1, A7C2, A7C3, A7C4, A7C5, A7C6, A7C7, A7C8, A7C9, A7C10, A7C11, A7C12, A7C13, A7C14, A7C15, A7C16, A7C17, A7C18, A7C19, A7C20, A7C21, A8C1, A8C2, A8C3, A8C4, A8C5, A8C6, A8C7, A8C8, A8C9, A8C10, A8C11, A8C12, A8C13, A8C14, A8C15, A8C16, A8C17, A8C18, A8C19, A8C20, A8C21, A9C1, A9C2, A9C3, A9C4, A9C5, A9C6, A9C7, A9C8, A9C9, A9C10, A9C11, A9C12, A9C13, A9C14, A9C15, A9C16, A9C17, A9C18, A9C19, A9C20, A9C21, A10C1, A10C2, A10C3, A10C4, A1005, A1006, A1007, A1008, A10C9, A10C10, A10C11, A10C12, A10C13, A10C14, A10C15, A10C16, A10C17, A10C18, A10C19, A10C20, A10C21, A11C1, A11C2, A11C3, A11C4, A1105, A1106, A11C7, A11C8, A11C9, A11C10, A11C11, A11C12, A11C13, A11C14, A11C15, A11C16, A11C17, A11C18, A11C19, A11C20, A11C21, B1C1, B1C2, B1C3, B1C4, B1C5, B1C6, B1C7, B1C8, B1C9, B1C10, B1C11, B1C12, B1C13, B1C14, B1C15, B1C16, B1C17, B1C18, B1C19, B1C20, B1C21, B2C1, B2C2, B2C3, B2C4, B2C5, B2C6, B2C7, B2C8, B2C9, B2C10, B2C11, B2C12, B2C13, B2C14, B2C15, B2C16, B2C17, B2C18, B2C19, B2C20, B2C21, B3C1, B3C2, B3C3, B3C4, B3C5, B3C6, B3C7, B3C8, B3C9, B3C10, B3C11, B3C12, B3C13, B3C14, B3C15, B3C16, B3C17, B3C18, B3C19, B3C20, B3C21, B4C1, B4C2, B4C3, B4C4, B4C5, B4C6, B4C7, B4C8, B4C9, B4C10, B4C11, B4C12, B4C13, B4C14, B4C15, B4C16, B4C17, B4C18, B4C19, B4C20, B4C21, B5C1, B5C2, B5C3, B5C4, B5C5, B5C6, B5C7, B5C8, B5C9, B5C10, B5C11, B5C12, B5C13, B5C14, B5C15, B5C16, B5C17, B5C18, B5C19, B5C20, B5C21, B6C1, B6C2, B6C3, B6C4, B6C5, B6C6, B6C7, B6C8, B6C9, B6C10, B6C11, B6C12, B6C13, B6C14, B6C15, B6C16, B6C17, B6C18, B6C19, B6C20, B6C21, B7C1, B7C2, B7C3, B7C4, B7C5, B7C6, B7C7, B7C8, B7C9, B7C10, B7C11, B7C12, B7C13, B7C14, B7C15, B7C16, B7C17, B7C18, B7C19, B7C20, B7C21, B8C1, B8C2, B8C3, B8C4, B8C5, B8C6, B8C7, B8C8, B8C9, B8C10, B8C11, B8C12, B8C13, B8C14, B8C15, B8C16, B8C17, B8C18, B8C19, B8C20, B8C21, B9C1, B9C2, B9C3, B9C4, B9C5, B9C6, B9C7, B9C8, B9C9, B9C10, B9C11, B9C12, B9C13, B9C14, B9C15, B9C16, B9C17, B9C18, B9C19, B9C20, B9C21, B10C1, B10C2, B10C3, B10C4, B10C5, B10C6, B10C7, B10C8, B10C9, B10C10, B10C11, B10C12, B10C13, B10C14, B10C15, B10C16, B10C17, B10C18, B10C19, B10C20, B10C21, B11C1, B11C2, B11C3, B11C4, B11C5, B11C6, B11C7, B11C8, B11C9, B11C10, B11C11, B11C12, B11C13, B11C14, B11C15, B11C16, B11C17, B11C18, B11C19, B11C20, B11C21, B12C1, B12C2, B12C3, B12C4, B12C5, B12C6, B12C7, B12C8, B12C9, B12C10, B12C11, B12C12, B12C13, B12C14, B12C15, B12C16, B12C17, B12C18, B12C19, B12C20, B12C21, A1B1C1, A1B1C2, A1B1C3, A1B1C4, A1B1C5, A1B1C6, A1B1C7, A1B1C8, A1B1C9, A1B1C10, A1B1C11, A1B1C12, A1B1C13, A1B1C14, A1B1C15, A1B1C16, A1B1C17, A1B1C18, A1B1C19, A1B1C20, A1B1C21, A1B2C1, A1B2C2, A1B2C3, A1B2C4, A1B2C5, A1B2C6, A1B2C7, A1B2C8, A1B2C9, A1B2C10, A1B2C11, A1B2C12, A1B2C13, A1B2C14, A1B2C15, A1B2C16, A1B2C17, A1B2C18, A1B2C19, A1B2C20, A1B2C21, A1B3C1, A1B3C2, A1B3C3, A1B3C4, A1B3C5, A1B3C6, A1B3C7, A1B3C8, A1B3C9, A1B3C10, A1B3C11, A1B3C12, A1B3C13, A1B3C14, A1B3C15, A1B3C16, A1B3C17, A1B3C18, A1B3C19, A1B3C20, A1B3C21, A1B4C1, A1B4C2, A1B4C3, A1B4C4, A1B4C5, A1B4C6, A1B4C7, A1B4C8, A1B4C9, A1B4C10, A1B4C11, A1B4C12, A1B4C13, A1B4C14, A1B4C15, A1B4C16, A1B4C17, A1B4C18, A1B4C19, A1B4C20, A1B4C21, A1B5C1, A1B5C2, A1B5C3, A1B5C4, A1B5C5, A1B5C6, A1B5C7, A1B5C8, A1B5C9, A1B5C10, A1B5C11, A1B5C12, A1B5C13, A1B5C14, A1B5C15, A1B5C16, A1B5C17, A1B5C18, A1B5C19, A1B5C20, A1B5C21, A1B6C1, A1B6C2, A1B6C3, A1B6C4, A1B6C5, A1B6C6, A1B6C7, A1B6C8, A1B6C9, A1B6C10, A1B6C11, A1B6C12, A1B6C13, A1B6C14, A1B6C15, A1B6C16, A1B6C17, A1B6C18, A1B6C19, A1B6C20, A1B6C21, A1B7C1, A1B7C2, A1B7C3, A1B7C4, A1B7C5, A1B7C6, A1B7C7, A1B7C8, A1B7C9, A1B7C10, A1B7C11, A1B7C12, A1B7C13, A1B7C14, A1B7C15, A1B7C16, A1B7C17, A1B7C18, A1B7C19, A1B7C20, A1B7C21, A1B8C1, A1B8C2, A1B8C3, A1B8C4, A1B8C5, A1B8C6, A1B8C7, A1B8C8, A1B8C9, A1B8C10, A1B8C11, A1B8C12, A1B8C13, A1B8C14, A1B8C15, A1B8C16, A1B8C17, A1B8C18, A1B8C19, A1B8C20, A1B8C21, A1B9C1, A1B9C2, A1B9C3, A1B9C4, A1B9C5, A1B9C6, A1B9C7, A1B9C8, A1B9C9, A1B9C10, A1B9C11, A1B9C12, A1B9C13, A1B9C14, A1B9C15, A1B9C16, A1B9C17, A1B9C18, A1B9C19, A1B9C20, A1B9C21, A1B10C1, A1B10C2, A1B10C3, A1B10C4, A1B10C5, A1B10C6, A1B10C7, A1B10C8, A1B10C9, A1B10C10, A1B10C11, A1B10C12, A1B10C13, A1B10C14, A1B10C15, A1B10C16, A1B10C17, A1B10C18, A1B10C19, A1B10C20, A1B10C21, A1B11C1, A1B11C2, A1B11C3, A1B11C4, A1B11C5, A1B11C6, A1B11C7, A1B11C8, A1B11C9, A1B11C10, A1B11C11, A1B11C12, A1B11C13, A1B11C14, A1B11C15, A1B11C16, A1B11C17, A1B11C18, A1B11C19, A1B11C20, A1B11C21, A1B12C1, A1B12C2, A1B12C3, A1B12C4, A1B12C5, A1B12C6, A1B12C7, A1B12C8, A1B12C9, A1B12C10, A1B12C11, A1B12C12, A1B12C13, A1B12C14, A1B12C15, A1B12C16, A1B12C17, A1B12C18, A1B12C19, A1B12C20, A1B12C21, A2B1C1, A2B1C2, A2B1C3, A2B1C4, A2B1C5, A2B1C6, A2B1C7, A2B1C8, A2B1C9, A2B1C10, A2B1C11, A2B1C12, A2B1C13, A2B1C14, A2B1C15, A2B1C16, A2B1C17, A2B1C18, A2B1C19, A2B1C20, A2B1C21, A2B2C1, A2B2C2, A2B2C3, A2B2C4, A2B2C5, A2B2C6, A2B2C7, A2B2C8, A2B2C9, A2B2C10, A2B2C11, A2B2C12, A2B2C13, A2B2C14, A2B2C15, A2B2C16, A2B2C17, A2B2C18, A2B2C19, A2B2C20, A2B2C21, A2B3C1, A2B3C2, A2B3C3, A2B3C4, A2B3C5, A2B3C6, A2B3C7, A2B3C8, A2B3C9, A2B3C10, A2B3C11, A2B3C12, A2B3C13, A2B3C14, A2B3C15, A2B3C16, A2B3C17, A2B3C18, A2B3C19, A2B3C20, A2B3C21, A2B4C1, A2B4C2, A2B4C3, A2B4C4, A2B4C5, A2B4C6, A2B4C7, A2B4C8, A2B4C9, A2B4C10, A2B4C11, A2B4C12, A2B4C13, A2B4C14, A2B4C15, A2B4C16, A2B4C17, A2B4C18, A2B4C19, A2B4C20, A2B4C21, A2B5C1, A2B5C2, A2B5C3, A2B5C4, A2B5C5, A2B5C6, A2B5C7, A2B5C8, A2B5C9, A2B5C10, A2B5C11, A2B5C12, A2B5C13, A2B5C14, A2B5C15, A2B5C16, A2B5C17, A2B5C18, A2B5C19, A2B5C20, A2B5C21, A2B6C1, A2B6C2, A2B6C3, A2B6C4, A2B6C5, A2B6C6, A2B6C7, A2B6C8, A2B6C9, A2B6C10, A2B6C11, A2B6C12, A2B6C13, A2B6C14, A2B6C15, A2B6C16, A2B6C17, A2B6C18, A2B6C19, A2B6C20, A2B6C21, A2B7C1, A2B7C2, A2B7C3, A2B7C4, A2B7C5, A2B7C6, A2B7C7, A2B7C8, A2B7C9, A2B7C10, A2B7C11, A2B7C12, A2B7C13, A2B7C14, A2B7C15, A2B7C16, A2B7C17, A2B7C18, A2B7C19, A2B7C20, A2B7C21, A2B8C1, A2B8C2, A2B8C3, A2B8C4, A2B8C5, A2B8C6, A2B8C7, A2B8C8, A2B8C9, A2B8C10, A2B8C11, A2B8C12, A2B8C13, A2B8C14, A2B8C15, A2B8C16, A2B8C17, A2B8C18, A2B8C19, A2B8C20, A2B8C21, A2B9C1, A2B9C2, A2B9C3, A2B9C4, A2B9C5, A2B9C6, A2B9C7, A2B9C8, A2B9C9, A2B9C10, A2B9C11, A2B9C12, A2B9C13, A2B9C14, A2B9C15, A2B9C16, A2B9C17, A2B9C18, A2B9C19, A2B9C20, A2B9C21, A2B10C1, A2B10C2, A2B10C3, A2B10C4, A2B10C5, A2B10C6, A2B10C7, A2B10C8, A2B10C9, A2B10C10, A2B10C11, A2B10C12, A2B10C13, A2B10C14, A2B10C15, A2B10C16, A2B10C17, A2B10C18, A2B10C19, A2B10C20, A2B10C21, A2B11C1, A2B11C2, A2B11C3, A2B11C4, A2B11C5, A2B11C6, A2B11C7, A2B11C8, A2B11C9, A2B11C10, A2B11C11, A2B11C12, A2B11C13, A2B11C14, A2B11C15, A2B11C16, A2B11C17, A2B11C18, A2B11C19, A2B11C20, A2B11C21, A2B12C1, A2B12C2, A2B12C3, A2B12C4, A2B12C5, A2B12C6, A2B12C7, A2B12C8, A2B12C9, A2B12C10, A2B12C11, A2B12C12, A2B12C13, A2B12C14, A2B12C15, A2B12C16, A2B12C17, A2B12C18, A2B12C19, A2B12C20, A2B12C21, A3B1C1, A3B1C2, A3B1C3, A3B1C4, A3B1C5, A3B1C6, A3B1C7, A3B1C8, A3B1C9, A3B1C10, A3B1C11, A3B1C12, A3B1C13, A3B1C14, A3B1C15, A3B1C16, A3B1C17, A3B1C18, A3B1C19, A3B1C20, A3B1C21, A3B2C1, A3B2C2, A3B2C3, A3B2C4, A3B2C5, A3B2C6, A3B2C7, A3B2C8, A3B2C9, A3B2C10, A3B2C11, A3B2C12, A3B2C13, A3B2C14, A3B2C15, A3B2C16, A3B2C17, A3B2C18, A3B2C19, A3B2C20, A3B2C21, A3B3C1, A3B3C2, A3B3C3, A3B3C4, A3B3C5, A3B3C6, A3B3C7, A3B3C8, A3B3C9, A3B3C10, A3B3C11, A3B3C12, A3B3C13, A3B3C14, A3B3C15, A3B3C16, A3B3C17, A3B3C18, A3B3C19, A3B3C20, A3B3C21, A3B4C1, A3B4C2, A3B4C3, A3B4C4, A3B4C5, A3B4C6, A3B4C7, A3B4C8, A3B4C9, A3B4C10, A3B4C11, A3B4C12, A3B4C13, A3B4C14, A3B4C15, A3B4C16, A3B4C17, A3B4C18, A3B4C19, A3B4C20, A3B4C21, A3B5C1, A3B5C2, A3B5C3, A3B5C4, A3B5C5, A3B5C6, A3B5C7, A3B5C8, A3B5C9, A3B5C10, A3B5C11, A3B5C12, A3B5C13, A3B5C14, A3B5C15, A3B5C16, A3B5C17, A3B5C18, A3B5C19, A3B5C20, A3B5C21, A3B6C1, A3B6C2, A3B6C3, A3B6C4, A3B6C5, A3B6C6, A3B6C7, A3B6C8, A3B6C9, A3B6C10, A3B6C11, A3B6C12, A3B6C13, A3B6C14, A3B6C15, A3B6C16, A3B6C17, A3B6C18, A3B6C19, A3B6C20, A3B6C21, A3B7C1, A3B7C2, A3B7C3, A3B7C4, A3B7C5, A3B7C6, A3B7C7, A3B7C8, A3B7C9, A3B7C10, A3B7C11, A3B7C12, A3B7C13, A3B7C14, A3B7C15, A3B7C16, A3B7C17, A3B7C18, A3B7C19, A3B7C20, A3B7C21, A3B8C1, A3B8C2, A3B8C3, A3B8C4, A3B8C5, A3B8C6, A3B8C7, A3B8C8, A3B8C9, A3B8C10, A3B8C11, A3B8C12, A3B8C13, A3B8C14, A3B8C15, A3B8C16, A3B8C17, A3B8C18, A3B8C19, A3B8C20, A3B8C21, A3B9C1, A3B9C2, A3B9C3, A3B9C4, A3B9C5, A3B9C6, A3B9C7, A3B9C8, A3B9C9, A3B9C10, A3B9C11, A3B9C12, A3B9C13, A3B9C14, A3B9C15, A3B9C16, A3B9C17, A3B9C18, A3B9C19, A3B9C20, A3B9C21, A3B10C1, A3B10C2, A3B10C3, A3B10C4, A3B10C5, A3B10C6, A3B10C7, A3B10C8, A3B10C9, A3B10C10, A3B10C11, A3B10C12, A3B10C13, A3B10C14, A3B10C15, A3B10C16, A3B10C17, A3B10C18, A3B10C19, A3B10C20, A3B10C21, A3B11C1, A3B11C2, A3B11C3, A3B11C4, A3B11C5, A3B11C6, A3B11C7, A3B11C8, A3B11C9, A3B11C10, A3B11C11, A3B11C12, A3B11C13, A3B11C14, A3B11C15, A3B11C16, A3B11C17, A3B11C18, A3B11C19, A3B11C20, A3B11C21, A3B12C1, A3B12C2, A3B12C3, A3B12C4, A3B12C5, A3B12C6, A3B12C7, A3B12C8, A3B12C9, A3B12C10, A3B12C11, A3B12C12, A3B12C13, A3B12C14, A3B12C15, A3B12C16, A3B12C17, A3B12C18, A3B12C19, A3B12C20, A3B12C21, A4B1C1, A4B1C2, A4B1C3, A4B1C4, A4B1C5, A4B1C6, A4B1C7, A4B1C8, A4B1C9, A4B1C10, A4B1C11, A4B1C12, A4B1C13, A4B1C14, A4B1C15, A4B1C16, A4B1C17, A4B1C18, A4B1C19, A4B1C20, A4B1C21, A4B2C1, A4B2C2, A4B2C3, A4B2C4, A4B2C5, A4B2C6, A4B2C7, A4B2C8, A4B2C9, A4B2C10, A4B2C11, A4B2C12, A4B2C13, A4B2C14, A4B2C15, A4B2C16, A4B2C17, A4B2C18, A4B2C19, A4B2C20, A4B2C21, A4B3C1, A4B3C2, A4B3C3, A4B3C4, A4B3C5, A4B3C6, A4B3C7, A4B3C8, A4B3C9, A4B3C10, A4B3C11, A4B3C12, A4B3C13, A4B3C14, A4B3C15, A4B3C16, A4B3C17, A4B3C18, A4B3C19, A4B3C20, A4B3C21, A4B4C1, A4B4C2, A4B4C3, A4B4C4, A4B4C5, A4B4C6, A4B4C7, A4B4C8, A4B4C9, A4B4C10, A4B4C11, A4B4C12, A4B4C13, A4B4C14, A4B4C15, A4B4C16, A4B4C17, A4B4C18, A4B4C19, A4B4C20, A4B4C21, A4B5C1, A4B5C2, A4B5C3, A4B5C4, A4B5C5, A4B5C6, A4B5C7, A4B5C8, A4B5C9, A4B5C10, A4B5C11, A4B5C12, A4B5C13, A4B5C14, A4B5C15, A4B5C16, A4B5C17, A4B5C18, A4B5C19, A4B5C20, A4B5C21, A4B6C1, A4B6C2, A4B6C3, A4B6C4, A4B6C5, A4B6C6, A4B6C7, A4B6C8, A4B6C9, A4B6C10, A4B6C11, A4B6C12, A4B6C13, A4B6C14, A4B6C15, A4B6C16, A4B6C17, A4B6C18, A4B6C19, A4B6C20, A4B6C21, A4B7C1, A4B7C2, A4B7C3, A4B7C4, A4B7C5, A4B7C6, A4B7C7, A4B7C8, A4B7C9, A4B7C10, A4B7C11, A4B7C12, A4B7C13, A4B7C14, A4B7C15, A4B7C16, A4B7C17, A4B7C18, A4B7C19, A4B7C20, A4B7C21, A4B8C1, A4B8C2, A4B8C3, A4B8C4, A4B8C5, A4B8C6, A4B8C7, A4B8C8, A4B8C9, A4B8C10, A4B8C11, A4B8C12, A4B8C13, A4B8C14, A4B8C15, A4B8C16, A4B8C17, A4B8C18, A4B8C19, A4B8C20, A4B8C21, A4B9C1, A4B9C2, A4B9C3, A4B9C4, A4B9C5, A4B9C6, A4B9C7, A4B9C8, A4B9C9, A4B9C10, A4B9C11, A4B9C12, A4B9C13, A4B9C14, A4B9C15, A4B9C16, A4B9C17, A4B9C18, A4B9C19, A4B9C20, A4B9C21, A4B10C1, A4B10C2, A4B10C3, A4B10C4, A4B10C5, A4B10C6, A4B10C7, A4B10C8, A4B10C9, A4B10C10, A4B10C11, A4B10C12, A4B10C13, A4B10C14, A4B10C15, A4B10C16, A4B10C17, A4B10C18, A4B10C19, A4B10C20, A4B10C21, A4B11C1, A4B11C2, A4B11C3, A4B11C4, A4B11C5, A4B11C6, A4B11C7, A4B11C8, A4B11C9, A4B11C10, A4B11C11, A4B11C12, A4B11C13, A4B11C14, A4B11C15, A4B11C16, A4B11C17, A4B11C18, A4B11C19, A4B11C20, A4B11C21, A4B12C1, A4B12C2, A4B12C3, A4B12C4, A4B12C5, A4B12C6, A4B12C7, A4B12C8, A4B12C9, A4B12C10, A4B12C11, A4B12C12, A4B12C13, A4B12C14, A4B12C15, A4B12C16, A4B12C17, A4B12C18, A4B12C19, A4B12C20, A4B12C21, A5B1C1, A5B1C2, A5B1C3, A5B1C4, A5B1C5, A5B1C6, A5B1C7, A5B1C8, A5B1C9, A5B1C10, A5B1C11, A5B1C12, A5B1C13, A5B1C14, A5B1C15, A5B1C16, A5B1C17, A5B1C18, A5B1C19, A5B1C20, A5B1C21, A5B2C1, A5B2C2, A5B2C3, A5B2C4, A5B2C5, A5B2C6, A5B2C7, A5B2C8, A5B2C9, A5B2C10, A5B2C11, A5B2C12, A5B2C13, A5B2C14, A5B2C15, A5B2C16, A5B2C17, A5B2C18, A5B2C19, A5B2C20, A5B2C21, A5B3C1, A5B3C2, A5B3C3, A5B3C4, A5B3C5, A5B3C6, A5B3C7, A5B3C8, A5B3C9, A5B3C10, A5B3C11, A5B3C12, A5B3C13, A5B3C14, A5B3C15, A5B3C16, A5B3C17, A5B3C18, A5B3C19, A5B3C20, A5B3C21, A5B4C1, A5B4C2, A5B4C3, A5B4C4, A5B4C5, A5B4C6, A5B4C7, A5B4C8, A5B4C9, A5B4C10, A5B4C11, A5B4C12, A5B4C13, A5B4C14, A5B4C15, A5B4C16, A5B4C17, A5B4C18, A5B4C19, A5B4C20, A5B4C21, A5B5C1, A5B5C2, A5B5C3, A5B5C4, A5B5C5, A5B5C6, A5B5C7, A5B5C8, A5B5C9, A5B5C10, A5B5C11, A5B5C12, A5B5C13, A5B5C14, A5B5C15, A5B5C16, A5B5C17, A5B5C18, A5B5C19, A5B5C20, A5B5C21, A5B6C1, A5B6C2, A5B6C3, A5B6C4, A5B6C5, A5B6C6, A5B6C7, A5B6C8, A5B6C9, A5B6C10, A5B6C11, A5B6C12, A5B6C13, A5B6C14, A5B6C15, A5B6C16, A5B6C17, A5B6C18, A5B6C19, A5B6C20, A5B6C21, A5B7C1, A5B7C2, A5B7C3, A5B7C4, A5B7C5, A5B7C6, A5B7C7, A5B7C8, A5B7C9, A5B7C10, A5B7C11, A5B7C12, A5B7C13, A5B7C14, A5B7C15, A5B7C16, A5B7C17, A5B7C18, A5B7C19, A5B7C20, A5B7C21, A5B8C1, A5B8C2, A5B8C3, A5B8C4, A5B8C5, A5B8C6, A5B8C7, A5B8C8, A5B8C9, A5B8C10, A5B8C11, A5B8C12, A5B8C13, A5B8C14, A5B8C15, A5B8C16, A5B8C17, A5B8C18, A5B8C19, A5B8C20, A5B8C21, A5B9C1, A5B9C2, A5B9C3, A5B9C4, A5B9C5, A5B9C6, A5B9C7, A5B9C8, A5B9C9, A5B9C10, A5B9C11, A5B9C12, A5B9C13, A5B9C14, A5B9C15, A5B9C16, A5B9C17, A5B9C18, A5B9C19, A5B9C20, A5B9C21, A5B10C1, A5B10C2, A5B10C3, A5B10C4, A5B10C5, A5B10C6, A5B10C7, A5B10C8, A5B10C9, A5B10C10, A5B10C11, A5B10C12, A5B10C13, A5B10C14, A5B10C15, A5B10C16, A5B10C17, A5B10C18, A5B10C19, A5B10C20, A5B10C21, A5B11C1, A5B11C2, A5B11C3, A5B11C4, A5B11C5, A5B11C6, A5B11C7, A5B11C8, A5B11C9, A5B11C10, A5B11C11, A5B11C12, A5B11C13, A5B11C14, A5B11C15, A5B11C16, A5B11C17, A5B11C18, A5B11C19, A5B11C20, A5B11C21, A5B12C1, A5B12C2, A5B12C3, A5B12C4, A5B12C5, A5B12C6, A5B12C7, A5B12C8, A5B12C9, A5B12C10, A5B12C11, A5B12C12, A5B12C13, A5B12C14, A5B12C15, A5B12C16, A5B12C17, A5B12C18, A5B12C19, A5B12C20, A5B12C21, A6B1C1, A6B1C2, A6B1C3, A6B1C4, A6B1C5, A6B1C6, A6B1C7, A6B1C8, A6B1C9, A6B1C10, A6B1C11, A6B1C12, A6B1C13, A6B1C14, A6B1C15, A6B1C16, A6B1C17, A6B1C18, A6B1C19, A6B1C20, A6B1C21, A6B2C1, A6B2C2, A6B2C3, A6B2C4, A6B2C5, A6B2C6, A6B2C7, A6B2C8, A6B2C9, A6B2C10, A6B2C11, A6B2C12, A6B2C13, A6B2C14, A6B2C15, A6B2C16, A6B2C17, A6B2C18, A6B2C19, A6B2C20, A6B2C21, A6B3C1, A6B3C2, A6B3C3, A6B3C4, A6B3C5, A6B3C6, A6B3C7, A6B3C8, A6B3C9, A6B3C10, A6B3C11, A6B3C12, A6B3C13, A6B3C14, A6B3C15, A6B3C16, A6B3C17, A6B3C18, A6B3C19, A6B3C20, A6B3C21, A6B4C1, A6B4C2, A6B4C3, A6B4C4, A6B4C5, A6B4C6, A6B4C7, A6B4C8, A6B4C9, A6B4C10, A6B4C11, A6B4C12, A6B4C13, A6B4C14, A6B4C15, A6B4C16, A6B4C17, A6B4C18, A6B4C19, A6B4C20, A6B4C21, A6B5C1, A6B5C2, A6B5C3, A6B5C4, A6B5C5, A6B5C6, A6B5C7, A6B5C8, A6B5C9, A6B5C10, A6B5C11, A6B5C12, A6B5C13, A6B5C14, A6B5C15, A6B5C16, A6B5C17, A6B5C18, A6B5C19, A6B5C20, A6B5C21, A6B6C1, A6B6C2, A6B6C3, A6B6C4, A6B6C5, A6B6C6, A6B6C7, A6B6C8, A6B6C9, A6B6C10, A6B6C11, A6B6C12, A6B6C13, A6B6C14, A6B6C15, A6B6C16, A6B6C17, A6B6C18, A6B6C19, A6B6C20, A6B6C21, A6B7C1, A6B7C2, A6B7C3, A6B7C4, A6B7C5, A6B7C6, A6B7C7, A6B7C8, A6B7C9, A6B7C10, A6B7C11, A6B7C12, A6B7C13, A6B7C14, A6B7C15, A6B7C16, A6B7C17, A6B7C18, A6B7C19, A6B7C20, A6B7C21, A6B8C1, A6B8C2, A6B8C3, A6B8C4, A6B8C5, A6B8C6, A6B8C7, A6B8C8, A6B8C9, A6B8C10, A6B8C11, A6B8C12, A6B8C13, A6B8C14, A6B8C15, A6B8C16, A6B8C17, A6B8C18, A6B8C19, A6B8C20, A6B8C21, A6B9C1, A6B9C2, A6B9C3, A6B9C4, A6B9C5, A6B9C6, A6B9C7, A6B9C8, A6B9C9, A6B9C10, A6B9C11, A6B9C12, A6B9C13, A6B9C14, A6B9C15, A6B9C16, A6B9C17, A6B9C18, A6B9C19, A6B9C20, A6B9C21, A6B10C1, A6B10C2, A6B10C3, A6B10C4, A6B10C5, A6B10C6, A6B10C7, A6B10C8, A6B10C9, A6B10C10, A6B10C11, A6B10C12, A6B10C13, A6B10C14, A6B10C15, A6B10C16, A6B10C17, A6B10C18, A6B10C19, A6B10C20, A6B10C21, A6B11C1, A6B11C2, A6B11C3, A6B11C4, A6B11C5, A6B11C6, A6B11C7, A6B11C8, A6B11C9, A6B11C10, A6B11C11, A6B11C12, A6B11C13, A6B11C14, A6B11C15, A6B11C16, A6B11C17, A6B11C18, A6B11C19, A6B11C20, A6B11C21, A6B12C1, A6B12C2, A6B12C3, A6B12C4, A6B12C5, A6B12C6, A6B12C7, A6B12C8, A6B12C9, A6B12C10, A6B12C11, A6B12C12, A6B12C13, A6B12C14, A6B12C15, A6B12C16, A6B12C17, A6B12C18, A6B12C19, A6B12C20, A6B12C21, A7B1C1, A7B1C2, A7B1C3, A7B1C4, A7B1C5, A7B1C6, A7B1C7, A7B1C8, A7B1C9, A7B1C10, A7B1C11, A7B1C12, A7B1C13, A7B1C14, A7B1C15, A7B1C16, A7B1C17, A7B1C18, A7B1C19, A7B1C20, A7B1C21, A7B2C1, A7B2C2, A7B2C3, A7B2C4, A7B2C5, A7B2C6, A7B2C7, A7B2C8, A7B2C9, A7B2C10, A7B2C11, A7B2C12, A7B2C13, A7B2C14, A7B2C15, 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A9B11C2, A9B11C3, A9B11C4, A9B11C5, A9B11C6, A9B11C7, A9B11C8, A9B11C9, A9B11C10, A9B11C11, A9B11C12, A9B11C13, A9B11C14, A9B11C15, A9B11C16, A9B11C17, A9B11C18, A9B11C19, A9B11C20, A9B11C21, A9B12C1, A9B12C2, A9B12C3, A9B12C4, A9B12C5, A9B12C6, A9B12C7, A9B12C8, A9B12C9, A9B12C10, A9B12C11, A9B12C12, A9B12C13, A9B12C14, A9B12C15, A9B12C16, A9B12C17, A9B12C18, A9B12C19, A9B12C20, A9B12C21, A10B1C1, A10B1C2, A10B1C3, A10B1C4, A10B1C5, A10B1C6, A10B1C7, A10B1C8, A10B1C9, A10B1C10, A10B1C11, A10B1C12, A10B1C13, A10B1C14, A10B1C15, A10B1C16, A10B1C17, A10B1C18, A10B1C19, A10B1C20, A10B1C21, A10B2C1, A10B2C2, A10B2C3, A10B2C4, A10B2C5, A10B2C6, A10B2C7, A10B2C8, A10B2C9, A10B2C10, A10B2C11, A10B2C12, A10B2C13, A10B2C14, A10B2C15, A10B2C16, A10B2C17, A10B2C18, A10B2C19, A10B2C20, A10B2C21, A10B3C1, A10B3C2, A10B3C3, A10B3C4, A10B3C5, A10B3C6, A10B3C7, A10B3C8, A10B3C9, A10B3C10, A10B3C11, A10B3C12, A10B3C13, A10B3C14, A10B3C15, A10B3C16, A10B3C17, A10B3C18, A10B3C19, A10B3C20, A10B3C21, A10B4C1, A10B4C2, A10B4C3, A10B4C4, A10B4C5, A10B4C6, A10B4C7, A10B4C8, A10B4C9, A10B4C10, A10B4C11, A10B4C12, A10B4C13, A10B4C14, A10B4C15, A10B4C16, A10B4C17, A10B4C18, A10B4C19, A10B4C20, A10B4C21, A10B5C1, A10B5C2, A10B5C3, A10B5C4, A10B5C5, A10B5C6, A10B5C7, A10B5C8, A10B5C9, A10B5C10, A10B5C11, A10B5C12, A10B5C13, A10B5C14, A10B5C15, A10B5C16, A10B5C17, A10B5C18, A10B5C19, A10B5C20, A10B5C21, A10B6C1, A10B6C2, A10B6C3, A10B6C4, A10B6C5, A10B6C6, A10B6C7, A10B6C8, A10B6C9, A10B6C10, A10B6C11, A10B6C12, A10B6C13, A10B6C14, A10B6C15, A10B6C16, A10B6C17, A10B6C18, A10B6C19, A10B6C20, A10B6C21, A10B7C1, A10B7C2, A10B7C3, A10B7C4, A10B7C5, A10B7C6, A10B7C7, A10B7C8, A10B7C9, A10B7C10, A10B7C11, A10B7C12, A10B7C13, A10B7C14, A10B7C15, A10B7C16, A10B7C17, A10B7C18, A10B7C19, A10B7C20, A10B7C21, A10B8C1, A10B8C2, A10B8C3, A10B8C4, A10B8C5, A10B8C6, A10B8C7, A10B8C8, A10B8C9, A10B8C10, A10B8C11, A10B8C12, A10B8C13, A10B8C14, A10B8C15, A10B8C16, A10B8C17, A10B8C18, A10B8C19, A10B8C20, A10B8C21, A10B9C1, A10B9C2, A10B9C3, A10B9C4, A10B9C5, A10B9C6, A10B9C7, A10B9C8, A10B9C9, A10B9C10, A10B9C11, A10B9C12, A10B9C13, A10B9C14, A10B9C15, A10B9C16, A10B9C17, A10B9C18, A10B9C19, A10B9C20, A10B9C21, A10B10C1, A10B10C2, A10B10C3, A10B10C4, A10B10C5, A10B10C6, A10B10C7, A10B10C8, A10B10C9, A10B10C10, A10B10C11, A10B10C12, A10B10C13, A10B10C14, A10B10C15, A10B10C16, A10B10C17, A10B10C18, A10B10C19, A10B10C20, A10B10C21, A10B11C1, A10B11C2, A10B11C3, A10B11C4, A10B11C5, A10B11C6, A10B11C7, A10B11C8, A10B11C9, A10B11C10, A10B11C11, A10B11C12, A10B11C13, A10B11C14, A10B11C15, A10B11C16, A10B11C17, A10B11C18, A10B11C19, A10B11C20, A10B11C21, A10B12C1, A10B12C2, A10B12C3, A10B12C4, A10B12C5, A10B12C6, A10B12C7, A10B12C8, A10B12C9, A10B12C10, A10B12C11, A10B12C12, A10B12C13, A10B12C14, A10B12C15, A10B12C16, A10B12C17, A10B12C18, A10B12C19, A10B12C20, A10B12C21, A11B1C1, A11B1C2, A11B1C3, A11B1C4, A11B1C5, A11B1C6, A11B1C7, A11B1C8, A11B1C9, A11B1C10, A11B1C11, A11B1C12, A11B1C13, A11B1C14, A11B1C15, A11B1C16, A11B1C17, A11B1C18, A11B1C19, A11B1C20, A11B1C21, A11B2C1, A11B2C2, A11B2C3, A11B2C4, A11B2C5, A11B2C6, A11B2C7, A11B2C8, A11B2C9, A11B2C10, A11B2C11, A11B2C12, A11B2C13, A11B2C14, A11B2C15, A11B2C16, A11B2C17, A11B2C18, A11B2C19, A11B2C20, A11B2C21, A11B3C1, A11B3C2, A11B3C3, A11B3C4, A11B3C5, A11B3C6, A11B3C7, A11B3C8, A11B3C9, A11B3C10, A11B3C11, A11B3C12, A11B3C13, A11B3C14, A11B3C15, A11B3C16, A11B3C17, A11B3C18, A11B3C19, A11B3C20, A11B3C21, A11B4C1, A11B4C2, A11B4C3, A11B4C4, A11B4C5, A11B4C6, A11B4C7, A11B4C8, A11B4C9, A11B4C10, A11B4C11, A11B4C12, A11B4C13, A11B4C14, A11B4C15, A11B4C16, A11B4C17, A11B4C18, A11B4C19, A11B4C20, A11B4C21, A11B5C1, A11B5C2, A11B5C3, A11B5C4, A11B5C5, A11B5C6, A11B5C7, A11B5C8, A11B5C9, A11B5C10, A11B5C11, A11B5C12, A11B5C13, A11B5C14, A11B5C15, A11B5C16, A11B5C17, A11B5C18, A11B5C19, A11B5C20, A11B5C21, A11B6C1, A11B6C2, A11B6C3, A11B6C4, A11B6C5, A11B6C6, A11B6C7, A11B6C8, A11B6C9, A11B6C10, A11B6C11, A11B6C12, A11B6C13, A11B6C14, A11B6C15, A11B6C16, A11B6C17, A11B6C18, A11B6C19, A11B6C20, A11B6C21, A11B7C1, A11B7C2, A11B7C3, A11B7C4, A11B7C5, A11B7C6, A11B7C7, A11B7C8, A11B7C9, A11B7C10, A11B7C11, A11B7C12, A11B7C13, A11B7C14, A11B7C15, A11B7C16, A11B7C17, A11B7C18, A11B7C19, A11B7C20, A11B7C21, A11B8C1, A11B8C2, A11B8C3, A11B8C4, A11B8C5, A11B8C6, A11B8C7, A11B8C8, A11B8C9, A11B8C10, A11B8C11, A11B8C12, A11B8C13, A11B8C14, A11B8C15, A11B8C16, A11B8C17, A11B8C18, A11B8C19, A11B8C20, A11B8C21, A11B9C1, A11B9C2, A11B9C3, A11B9C4, A11B9C5, A11B9C6, A11B9C7, A11B9C8, A11B9C9, A11B9C10, A11B9C11, A11B9C12, A11B9C13, A11B9C14, A11B9C15, A11B9C16, A11B9C17, A11B9C18, A11B9C19, A11B9C20, A11B9C21, A11B10C1, A11B10C2, A11B10C3, A11B10C4, A11B10C5, A11B10C6, A11B10C7, A11B10C8, A11B10C9, A11B10C10, A11B10C11, A11B10C12, A11B10C13, A11B10C14, A11B10C15, A11B10C16, A11B10C17, A11B10C18, A11B10C19, A11B10C20, A11B10C21, A11B11C1, A11B11C2, A11B11C3, A11B11C4, A11B11C5, A11B11C6, A11B11C7, A11B11C8, A11B11C9, A11B11C10, A11B11C11, A11B11C12, A11B11C13, A11B11C14, A11B11C15, A11B11C16, A11B11C17, A11B11C18, A11B11C19, A11B11C20, A11B11C21, A11B12C1, A11B12C2, A11B12C3, A11B12C4, A11B12C5, A11B12C6, A11B12C7, A11B12C8, A11B12C9, A11B12C10, A11B12C11, A11B12C12, A11B12C13, A11B12C14, A11B12C15, A11B12C16, A11B12C17, A11B12C18, A11B12C19, A11B12C20, or A11B12C21.

STING Agonists

In an embodiment, the combination includes a STING agonist. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein e.g., a solid tumor (e.g., a breast cancer, a squamous cell carcinoma, a melanoma, an ovarian cancer, a fallopian tube carcinoma, a peritoneal carcinoma, a soft tissue sarcoma, a melanoma, a breast cancer, an esophageal cancer, a head and neck cancer, an endometrial cancer, a cervical cancer, or a basal cell carcinoma), e.g., a hematologic malignancy (e.g., a leukemia (e.g., a chronic lymphocytic leukemia (CLL), or a lymphoma (e.g., a marginal zone B-cell lymphoma, a small lymphocytic lymphoma, a follicular lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma)).

In some embodiments, the STING agonist is cyclic dinucleotide, e.g., a cyclic dinucleotide comprising purine or pyrimidine nucleobases (e.g., adenosine, guanine, uracil, thymine, or cytosine nucleobases). In some embodiments, the nucleobases of the cyclic dinucleotide comprise the same nucleobase or different nucleobases.

In some embodiments, the STING agonist comprises an adenosine or a guanosine nucleobase. In some embodiments, the STING agonist comprises one adenosine nucleobase and one guanosine nucleobase. In some embodiments, the STING agonist comprises two adenosine nucleobases or two guanosine nucleobases.

In some embodiments, the STING agonist comprises a modified cyclic dinucleotide, e.g., comprising a modified nucleobase, a modified ribose, or a modified phosphate linkage. In some embodiments, the modified cyclic dinucleotide comprises a modified phosphate linkage, e.g., a thiophosphate.

In some embodiments, the STING agonist comprises a cyclic dinucleotide (e.g., a modified cyclic dinucleotide) with 2′,5′ or 3′,5′ phosphate linkages. In some embodiments, the STING agonist comprises a cyclic dinucleotide (e.g., a modified cyclic dinucleotide) with Rp or Sp stereochemistry around the phosphate linkages.

In some embodiments, the STING agonist is Rp,Rp dithio 2′,3′ c-di-AMP (e.g., Rp,Rp-dithio c-[A(2′,5′)pA(3′,5′)p]), or a cyclic dinucleotide analog thereof. In some embodiments, the STING agonist is a compound depicted in U.S. Patent Publication No. US2015/0056224 (e.g., a compound in FIG. 2c , e.g., compound 21 or compound 22). In some embodiments, the STING agonist is c-[G(2′,5′)pG(3′,5′)p], a dithio ribose O-substituted derivative thereof, or a compound depicted in FIG. 4 of PCT Publication Nos. WO 2014/189805 and WO 2014/189806. In some embodiments, the STING agonist is c-[A(2′,5′)pA(3′,5′)p] or a dithio ribose 0-substitued derivative thereof, or is a compound depicted in FIG. 5 of PCT Publication Nos. WO 2014/189805 and WO 2014/189806. In some embodiments, the STING agonist is c-[G(2′,5′)pA(3′,5′)p], or a dithio ribose 0-substitued derivative thereof, or is a compound depicted in FIG. 5 of PCT Publication Nos. WO 2014/189805 and WO 2014/189806. In some embodiments, the STING agonist is 2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p] (2′-O-propargyl-ML-CDA) or a compound depicted in FIG. 7 of PCT Publication No. WO 2014/189806.

Other exemplary STING agonists are disclosed, e.g., in PCT Publication Nos. WO 2014/189805 and WO 2014/189806, and U.S. Publication No. 2015/0056225.

TLR Agonists

In an embodiment, a combination described herein includes a Toll-like receptor (TLR) agonist. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a breast cancer, a squamous cell carcinoma, a melanoma, an ovarian cancer, a fallopian tube carcinoma, a peritoneal carcinoma, a soft tissue sarcoma, a melanoma, a breast cancer, an esophageal cancer, a head and neck cancer, an endometrial cancer, a cervical cancer, or a basal cell carcinoma), e.g., a hematologic malignancy (e.g., a leukemia (e.g., a chronic lymphocytic leukemia (CLL), or a lymphoma (e.g., a marginal zone B-cell lymphoma, a small lymphocytic lymphoma, a follicular lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma)).

TLRs are a family of pattern recognition receptors that were initially identified as sensors of the innate immune system that recognize microbial pathogens. In humans, the TLRs include TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, TLR-9, and TLR-10. TLR-1, -2, -4, -5, and -6, are expressed on the surface of cells and TLR-3, -7/8, and -9 are expressed with the ER compartment. Human dendritic cell subsets can be identified on the basis of distinct TLR expression patterns. The myeloid or “conventional” subset of human dendritic cells express TLRs 1-8 and the plasmacytoid subset of dendritic cells express only TLR-7 and TLR-9. Ligand binding to TLRs invokes a cascade of intra-cellular signaling pathways that induce the production of factors involved in inflammation and immunity. Upon stimulation, the myeloid subset and the plasmacytoid subset of human dendritic cells result in antigen-specific CD4+ and CD8+ T cell priming and activation of NK cells and T-cells, respectively.

In some embodiments, the TLR agonist is chosen from one or more of a TLR-1 agonist, a TLR-2 agonist, a TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a TLR-6 agonist, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist, a TLR-10 agonist, a TLR-1/2 agonist, a TLR-2/6 agonist, or a TLR-7/8 agonist. In one embodiment, the TLR agonist is a TLR7 agonist.

In some embodiments, the TLR agonist is imiquimod or 3-(2-Methylpropyl)-3,5,8-triazatricyclo[7.4.0.02,6]trideca-1(9),2(6),4,7,10,12-hexaen-7-amine. Imiquimod or 3-(2-Methylpropyl)-3,5,8-triazatricyclo[7.4.0.02,6]trideca-1(9),2(6),4,7,10,12-hexaen-7-amine can bind to and activate TLR-7 and/or TLR-8.

In some embodiments, the TLR agonist is 852A. 852A is disclosed, e.g., in Inglefield et al. J Interferon Cytokine Res. 2008; 28(4):253-63. 852A can bind to and activate TLR-7 and/or TLR-8.

In some embodiments, the TLR agonist is Bacille Calmette-Guérin (BCG). BCG can bind to and activate TLR-9.

In some embodiments, the TLR agonist is EMD 120108. EMD 120108 is a synthetic oligonucleotide containing phosphorothioate oligodeoxynucleotide. EMD 1201081 can bind to and activate TLR-9, e.g, in monocytes/macrophages, plasmacytoid dendritic cells (DCs) and B cells, initiating immune signaling pathways, activating B cells and inducing T-helper cell cytokine production.

In some embodiments, the TLR agonist is IMO-2055. IMO-2055 is a synthetic oligonucleotide containing unmethylated CpG dinucleotides. Mimicking unmethylated CpG sequences in bacterial DNA, IMO-2055 can bind to and activate TLR-9, e.g., in monocytes/macrophages, plasmacytoid dendritic cells (DCs) and B cells, initiating immune signaling pathways and activating B cells and DCs and inducing T-helper cell cytokine production.

Other exemplary TLR agonists that can be used in the combination include, e.g., TLR-1/2 agonists (e.g., Pam3Cys), TLR-2 agonists (e.g., CFA, MALP2, Pam2Cys, FSL-1, or Hib-OMPC), TLR-3 agonists (e.g., polyribosinic:polyribocytidic acid (Poly I:C), polyadenosine-polyuridylic acid (poly AU), polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (Hiltonol®)), TLR-4 agonists (e.g., monophosphoryl lipid A (MPL), LPS, sialyl-Tn (STn)), TLR-5 agonists (e.g., bacterial flagellin), TLR-7 agonists (e.g., imiquimod), TLR-7/8 agonists (e.g., resiquimod or loxoribine), and TLR-9 agonists (e.g., unmethylated CpG dinucleotide (CpG-ODN)).

In another embodiment, the TLR agonist is used in combination with a GITR agonist, e.g., as described in WO2004060319, and International Publication No.: WO2014012479.

VEGFR Inhibitors

In one embodiment, a combination described herein includes a vascular endothelial growth factor (VEGF) receptor inhibitor (e.g., an inhibitor of one or more of VEGFR (e.g., VEGFR-1, VEGFR-2, VEGFR-3) or VEGF). In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a melanoma, a breast cancer, a colon cancer, an esophageal cancer, a gastrointestinal stromal tumor (GIST), a kidney cancer (e.g., a renal cell cancer), a liver cancer, a non-small cell lung cancer (NSCLC), an ovarian cancer, a pancreatic cancer, a prostate cancer, or a stomach cancer), e.g., a hematologic malignancy (e.g., a lymphoma).

In some embodiments, the VEGFR inhibitor is vatalanib succinate (Compound A47) or a compound disclosed in EP 296122.

In some embodiment, the VEGFR inhibitor is an inhibitor of one or more of VEGFR-2, PDGFRbeta, KIT or Raf kinase C, 1-methyl-5-((2-(5-(trifluoromethyl)-1H-imidazol-2-yl)pyridin-4-yl)oxy)-N-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-2-amine (Compound A37) or a compound disclosed in PCT Publication No. WO 2007/030377.

Other exemplary VEGFR pathway inhibitors that can be used in the combinations disclosed herein include, e.g., bevacizumab (AVASTIN®), axitinib (INLYTA®); brivanib alaninate (BMS-582664, (S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate); sorafenib (NEXAVAR®); pazopanib (VOTRIENT®); sunitinib malate (SUTENT®); cediranib (AZD2171, CAS 288383-20-1); vargatef (BIBF1120, CAS 928326-83-4); Foretinib (GSK1363089); telatinib (BAY57-9352, CAS 332012-40-5); apatinib (YN968D1, CAS 811803-05-1); imatinib (GLEEVEC®); ponatinib (AP24534, CAS 943319-70-8); tivozanib (AV951, CAS 475108-18-0); regorafenib (BAY73-4506, CAS 755037-03-7); vatalanib dihydrochloride (PTK787, CAS 212141-51-0); brivanib (BMS-540215, CAS 649735-46-6); vandetanib (CAPRELSA® or AZD6474); motesanib diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide, described in PCT Publication No. WO 02/066470); dovitinib dilactic acid (TKI258, CAS 852433-84-2); linfanib (ABT869, CAS 796967-16-3); cabozantinib (XL184, CAS 849217-68-1); lestaurtinib (CAS 111358-88-4); N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); 4-methyl-3-[[1-methyl-6-(3-pyridinyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide (BHG712, CAS 940310-85-0); aflibercept (EYLEA®), and endostatin (ENDOSTAR®).

Exemplary anti-VEGF antibodies that can be used in the combinations disclosed herein include, e.g., a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. (1997) Cancer Res. 57:4593-4599. In one embodiment, the anti-VEGF antibody is Bevacizumab (BV), also known as rhuMAb VEGF or AVASTIN®. It comprises mutated human IgG1 framework regions and antigen-binding complementarity-determining regions from the murine anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding of human VEGF to its receptors. Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. Pat. No. 6,884,879 issued Feb. 26, 2005. Additional antibodies include the G6 or B20 series antibodies (e.g., G6-31, B20-4.1), as described in PCT Publication No. WO2005/012359, PCT Publication No. WO2005/044853, the contents of these patent applications are expressly incorporated herein by reference. For additional antibodies see U.S. Pat. Nos. 7,060,269, 6,582,959, 6,703,020, 6,054,297, WO98/45332, WO 96/30046, WO94/10202, EP 0666868B1, U.S. Patent Application Publication Nos. 2006009360, 20050186208, 20030206899, 20030190317, 20030203409, and 20050112126; and Popkov et al, Journal of Immunological Methods 288: 149-164 (2004). Other antibodies include those that bind to a functional epitope on human VEGF comprising of residues F17, M18, D19, Y21, Y25, Q89, 191, K1 01, E1 03, and C104 or, alternatively, comprising residues F17, Y21, Q22, Y25, D63, 183 and Q89.

c-MET Inhibitors

In one embodiment, a combination described herein includes an inhibitor of c-MET. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a non-small cell lung cancer, a pancreatic cancer, a liver cancer, a thyroid cancer, a brain tumor (e.g., a glioblastoma), a kidney cancer (e.g., a renal cell carcinoma), a head and neck cancer (e.g., a head and neck squamous cell carcinoma).

In some embodiments, the c-MET inhibitor is Compound A17 or a compound described in U.S. Pat. Nos. 7,767,675 and 8,420,645). c-MET, a receptor tyrosine kinase overexpressed or mutated in many tumor cell types, plays key roles in tumor cell proliferation, survival, invasion, metastasis, and tumor angiogenesis. Inhibition of c-MET may induce cell death in tumor cells overexpressing c-MET protein or expressing constitutively activated c-MET protein.

In some embodiments, the c-MET inhibitor is JNJ-38877605. JNJ-38877605 is an orally available, small molecule inhibitor of c-Met. JNJ-38877605 selectively binds to c-MET, thereby inhibiting c-MET phosphorylation and disrupting c-Met signal transduction pathways.

In some embodiments, the c-Met inhibitor is AMG 208. AMG 208 is a selective small-molecule inhibitor of c-MET. AMG 208 inhibits the ligand-dependent and ligand-independent activation of c-MET, inhibiting its tyrosine kinase activity, which may result in cell growth inhibition in tumors that overexpress c-Met.

In some embodiments, the c-Met inhibitor is AMG 337. AMG 337 is an orally bioavailable inhibitor of c-Met. AMG 337 selectively binds to c-MET, thereby disrupting c-MET signal transduction pathways.

In some embodiments, the c-Met inhibitor is LY2801653. LY2801653 is an orally available, small molecule inhibitor of c-Met. LY2801653 selectively binds to c-MET, thereby inhibiting c-MET phosphorylation and disrupting c-Met signal transduction pathways.

In some embodiments, c-Met inhibitor is MSC2156119J. MSC2156119J is an orally bioavailable inhibitor of c-Met. MSC2156119J selectively binds to c-MET, which inhibits c-MET phosphorylation and disrupts c-Met-mediated signal transduction pathways.

In some embodiments, the c-MET inhibitor is capmatinib. Capmatinib is also known as INCB028060. Capmatinib is an orally bioavailable inhibitor of c-MET. Capmatinib selectively binds to c-Met, thereby inhibiting c-Met phosphorylation and disrupting c-Met signal transduction pathways.

In some embodiments, the c-MET inhibitor is crizotinib. Crizotinib is also known as PF-02341066. Crizotinib is an orally available aminopyridine-based inhibitor of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) and the c-Met/hepatocyte growth factor receptor (HGFR). Crizotinib, in an ATP-competitive manner, binds to and inhibits ALK kinase and ALK fusion proteins. In addition, crizotinib inhibits c-Met kinase, and disrupts the c-Met signaling pathway. Altogether, this agent inhibits tumor cell growth.

In some embodiments, the c-MET inhibitor is golvatinib. Golvatinib is an orally bioavailable dual kinase inhibitor of c-MET and VEGFR-2 with potential antineoplastic activity. Golvatinib binds to and inhibits the activities of both c-MET and VEGFR-2, which may inhibit tumor cell growth and survival of tumor cells that overexpress these receptor tyrosine kinases.

In some embodiments, the c-MET inhibitor is tivantinib. Tivantinib is also known as ARQ 197. Tivantinib is an orally bioavailable small molecule inhibitor of c-MET. Tivantinib binds to the c-MET protein and disrupts c-Met signal transduction pathways, which may induce cell death in tumor cells overexpressing c-MET protein or expressing consitutively activated c-Met protein.

TGFb Inhibitors

In one embodiment, a combination described herein includes a transforming growth factor beta (TGF-β) inhibitor. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a brain cancer (e.g., a glioma), a melanoma, a kidney cancer (e.g., a renal cell carcinoma), a pleural malignant mesothelioma (e.g., a relapsed pleural malignant mesothelioma), or a breast cancer (e.g., a metastatic breast cancer)).

In some embodiments, the TGF-β inhibitor is fresolimumab (CAS Registry Number: 948564-73-6). Fresolimumab is also known as GC1008. Fresolimumab is a human monoclonal antibody that binds to and inhibits TGF-beta isoforms 1, 2 and 3.

Fresolimumab is disclosed, e.g., in WO 2006/086469, U.S. Pat. No. 8,383,780, and U.S. Pat. No. 8,591,901.

In some embodiments, the TGF-β inhibitor is XOMA 089. XOMA 089 is also known as XPA.42.089. XOMA 089 is a fully human monoclonal antibody that specifically binds and neutralizes TGF-beta 1 and 2 ligands.

The heavy chain variable region of XOMA 089 has the amino acid sequence disclosed as SEQ ID NO: 6 in WO 2012/167143. The light chain variable region of XOMA 089 has the amino acid sequence disclosed as SEQ ID NO: 8 in WO 2012/167143.

IDO/TDO Inhibitors

In one embodiment, a combination described herein includes an inhibitor of indoleamine 2,3-dioxygenase (IDO) and/or tryptophan 2,3-dioxygenase (TDO). In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., melanoma, non-small cell lung cancer, colon cancer, squamous cell head and neck cancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, breast cancer (e.g., metastatic or HER2-negative breast cancer)), e.g., a hematologic malignancy (e.g., a lymphoma, e.g., a non-Hodgkin's lymphoma or a Hodgkin's lymphoma (e.g., a diffuse large B-cell lymphoma (DLBCL))).

In some embodiments, the IDO/TDO inhibitor is chosen from (4E)-4-[(3-chloro-4-fluoroanilino)-nitrosomethylidene]-1,2,5-oxadiazol-3-amine (also known as INCB24360), indoximod (1-methyl-D-tryptophan), or α-cyclohexyl-5H-Imidazo[5,1-a]isoindole-5-ethanol (also known as NLG919).

In some embodiments, the IDO/TDO inhibitor is epacadostat (CAS Registry Number: 1204669-58-8). Epacadostat is also known as INCB24360 or INCB024360 (Incyte). Epacadostat is a potent and selective indoleamine 2,3-dioxygenase (IDO1) inhibitor with IC50 of 10 nM, highly selective over other related enzymes such as IDO2 or tryptophan 2,3-dioxygenase (TDO).

In some embodiments, the IDO/TDO inhibitor is indoximod (New Link Genetics). Indoximod, the D isomer of 1-methyl-tryptophan, is an orally administered small-molecule indoleamine 2,3-dioxygenase (IDO) pathway inhibitor that disrupts the mechanisms by which tumors evade immune-mediated destruction.

In some embodiments, the IDO/TDO inhibitor is NLG919 (New Link Genetics). NLG919 is a potent IDO (indoleamine-(2,3)-dioxygenase) pathway inhibitor with Ki/EC50 of 7 nM/75 nM in cell-free assays.

In some embodiments, the IDO/TDO inhibitor is F001287 (Flexus/BMS). F001287 is a small molecule inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1).

A2AR Antagonists

In one embodiment, a combination described herein includes an adenosine A2a receptor (A2aR) antagonist (e.g., an inhibitor of A2aR pathway, e.g., an adenosine inhibitor, e.g., an inhibitor of A2aR or CD-73). In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein.

In some embodiments, the A2aR antagonist is istradefylline (CAS Registry Number: 155270-99-8). Istradefylline is also known as KW-6002 or 8-[(E)-2-(3,4-dimethoxyphenyl)vinyl]-1,3-diethyl-7-methyl-3,7-dihydro-1H-purine-2,6-dione. Istradefylline is disclosed, e.g., in LeWitt et al. (2008) Annals of Neurology 63 (3): 295-302).

In some embodiments, the A2aR antagonist is tozadenant (Biotie). Tozadenant is also known as SYN115 or 4-hydroxy-N-(4-methoxy-7-morpholin-4-yl-1,3-benzothiazol-2-yl)-4-methylpiperidine-1-carboxamide. Tozadenant blocks the effect of endogenous adenosine at the A2a receptors, resulting in the potentiation of the effect of dopamine at the D2 receptor and inhibition of the effect of glutamate at the mGluR5 receptor. e.g., In some embodiments, the A2aR antagonist is preladenant (CAS Registry Number: 377727-87-2). Preladenant is also known as SCH 420814 or 2-(2-Furanyl)-7-[2-[4-[4-(2-methoxyethoxy)phenyl]-1-piperazinyl]ethyl]7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine-5-amine. Preladenant was developed as a drug that acted as a potent and selective antagonist at the adenosine A2A receptor.

In some embodiments, the A2aR antagonist is vipadenan. Vipadenan is also known as BIIB014, V2006, or 3-[(4-amino-3-methylphenyl)methyl]-7-(furan-2-yl)triazolo[4,5-d]pyrimidin-5-amine. e.g., In some embodiments, the A2aR antagonist is PBF-509 (Palobiofarma). e.g., In some embodiments, the A2aR antagonist, e.g., PBF-509 is administered at a daily dose of about 80 mg, 160 mg, or 240 mg.

Other exemplary A2aR antagonists include, e.g., ATL-444, MSX-3, SCH-58261, SCH-412,348, SCH-442,416, VER-6623, VER-6947, VER-7835, CGS-15943, or ZM-241,385.

In some embodiments, the A2aR antagonist is an A2aR pathway antagonist (e.g., a CD-73 inhibitor, e.g., an anti-CD73 antibody) is MEDI9447. MEDI9447 is a monoclonal antibody specific for CD73. Targeting the extracellular production of adenosine by CD73 may reduce the immunosuppressive effects of adenosine. MEDI9447 was reported to have a range of activities, e.g., inhibition of CD73 ectonucleotidase activity, relief from AMP-mediated lymphocyte suppression, and inhibition of syngeneic tumor growth. MEDI9447 can drive changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment. These changes include, e.g., increases in CD8 effector cells and activated macrophages, as well as a reduction in the proportions of myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes.

Oncolytic Viruses

In some embodiments, a combination as described herein includes an oncolytic virus. In embodiments, oncolytic viruses are capable of selectively replicating in and triggering the death of or slowing the growth of a cancer cell. In some cases, oncolytic viruses have no effect or a minimal effect on non-cancer cells. An oncolytic virus includes but is not limited to an oncolytic adenovirus, oncolytic Herpes Simplex Viruses, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic Sinbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g., oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (VSV)).

In some embodiments, the oncolytic virus is a virus, e.g., recombinant oncolytic virus, described in US2010/0178684 A1, which is incorporated herein by reference in its entirety. In some embodiments, a recombinant oncolytic virus comprises a nucleic acid sequence (e.g., heterologous nucleic acid sequence) encoding an inhibitor of an immune or inflammatory response, e.g., as described in US2010/0178684 A1, incorporated herein by reference in its entirety. In embodiments, the recombinant oncolytic virus, e.g., oncolytic NDV, comprises a pro-apoptotic protein (e.g., apoptin), a cytokine (e.g., GM-CSF, CSF, interferon-gamma, interleukin-2 (IL-2), tumor necrosis factor-alpha), an immunoglobulin (e.g., an antibody against ED-B firbonectin), tumor associated antigen, a bispecific adapter protein (e.g., bispecific antibody or antibody fragment directed against NDV HN protein and a T cell co-stimulatory receptor, such as CD3 or CD28; or fusion protein between human IL-2 and single chain antibody directed against NDV HN protein). See, e.g., Zamarin et al. Future Microbiol. 7.3(2012):347-67, incorporated herein by reference in its entirety. In some embodiments, the oncolytic virus is a chimeric oncolytic NDV described in U.S. Pat. No. 8,591,881 B2, US 2012/0122185 A1, or US 2014/0271677 A1, each of which is incorporated herein by reference in their entireties.

In some embodiments, the oncolytic virus comprises a conditionally replicative adenovirus (CRAd), which is designed to replicate exclusively in cancer cells. See, e.g., Alemany et al. Nature Biotechnol. 18(2000):723-27. In some embodiments, an oncolytic adenovirus comprises one described in Table 1 on page 725 of Alemany et al., incorporated herein by reference in its entirety.

Exemplary oncolytic viruses include but are not limited to the following:

Group B Oncolytic Adenovirus (ColoAdl) (PsiOxus Therapeutics Ltd.) (see, e.g., Clinical Trial Identifier: NCT02053220);

ONCOS-102 (previously called CGTG-102), which is an adenovirus comprising granulocyte-macrophage colony stimulating factor (GM-CSF) (Oncos Therapeutics) (see, e.g., Clinical Trial Identifier: NCT01598129);

VCN-01, which is a genetically modified oncolytic human adenovirus encoding human PH20 hyaluronidase (VCN Biosciences, S.L.) (see, e.g., Clinical Trial Identifiers: NCT02045602 and NCT02045589);

Conditionally Replicative Adenovirus ICOVIR-5, which is a virus derived from wild-type human adenovirus serotype 5 (Had5) that has been modified to selectively replicate in cancer cells with a deregulated retinoblastoma/E2F pathway (Institut Catala d'Oncologia) (see, e.g., Clinical Trial Identifier: NCT01864759);

Celyvir, which comprises bone marrow-derived autologous mesenchymal stem cells (MSCs) infected with ICOVIR5, an oncolytic adenovirus (Hospital Infantil Universitario Niño Jesús, Madrid, Spain/Ramon Alemany) (see, e.g., Clinical Trial Identifier: NCT01844661);

CG0070, which is a conditionally replicating oncolytic serotype 5 adenovirus (Ad5) in which human E2F-1 promoter drives expression of the essential Ela viral genes, thereby restricting viral replication and cytotoxicity to Rb pathway-defective tumor cells (Cold Genesys, Inc.) (see, e.g., Clinical Trial Identifier: NCT02143804); or

DNX-2401 (formerly named Delta-24-RGD), which is an adenovirus that has been engineered to replicate selectively in retinoblastoma (Rb)-pathway deficient cells and to infect cells that express certain RGD-binding integrins more efficiently (Clinica Universidad de Navarra, Universidad de Navarra/DNAtrix, Inc.) (see, e.g., Clinical Trial Identifier: NCT01956734).

In some embodiments, an oncolytic virus described herein is administering by injection, e.g., subcutaneous, intra-arterial, intravenous, intramuscular, intrathecal, or intraperitoneal injection. In embodiments, an oncolytic virus described herein is administered intratumorally, transdermally, transmuco sally, orally, intranasally, or via pulmonary administration.

Vaccines, e.g., Scaffold Vaccines

In one embodiment, a combination described herein includes a vaccine, e.g., a scaffold vaccine. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein.

Cancer vaccines are disclosed, e.g., in PCT Publication Nos. WO 2007/070660 and WO 2012/167230, EP 1960009 B1, U.S. Pat. No. 8,067,237 and U.S. Pat. No. 8,932,583, and U.S. Publication No. US 2011/0020216. The components that can be used within cancer vaccines (e.g., implantable scaffold materials) are disclosed, e.g., in PCT Publication Nos. WO 2009/102465 and WO 2013/106852. Methods that can be used for administration of cancer vaccines are disclosed, e.g., in PCT Publication Nos. WO 2013/158673, WO 2012/048165, and WO 2012/149358.

In some embodiments, the cancer vaccine includes a macroporous scaffold comprising (i) cells or a cell recruitment composition, and (ii) a deployment signal capable of inducing or promoting migration of cells, and (iii) a bioactive composition coated or seeded onto/into the scaffold, which causes cells recruited into the scaffold be modified. Migration of the modified cells can be promoted by the open, interconnected macropores and the deployment signal.

In some embodiments, the cancer vaccine induces an endogenous immune response to a cancer target via administration of a porous scaffold bearing a recruitment composition and a target antigen composition, wherein an endogenous antigen presenting cell is recruited into the scaffold to encounter antigen and where said cell resides until a deployment signal induces egress to a lymph node tissue outside the scaffold, thereby stimulating an endogenous immune response to said cancer target.

In some embodiments, the cancer vaccine is used to remove a target cell from a mammal using a scaffold composition.

In some embodiments, an in situ cancer vaccine is generated via recruitment of cancer cells to an implanted scaffold and destruction of the cells using a cytotoxic agent.

In some embodiments, a cytosine-guanosine oligonucleotide (CpG-ODN) is used as a component of a scaffold, which can effectively reprogram and deploy dendritic cells recruited to the scaffold, and generate an effective anti-tumor response.

In some embodiments, polyinosine-polycytidylic acid (poly I:C) and/or CpG ODN are used to exert a synergistic effect on tumor inhibition.

In some embodiments, porous rods comprising an immune cell recruitment compound (e.g. GM-CSF) and an immune cell activation compound (e.g. CpG ODN), and optionally comprising an antigen such as a tumor lysate, are used, e.g., to elicit an immune response to a vaccine antigen. In some embodiments, pores that facilitate recruitment or release of cells are formed in situ within hydrogels following hydrogel injection. In some embodiments, injectable shape memory porous hydrogel polymer is used for administration.

In other embodiments, the combinations disclosed herein include a cancer or tumor vaccine. Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, tumor cells transfected to express the cytokine GM-CSF, DNA-based vaccines, RNA-based vaccines, and viral transduction-based vaccines. The cancer vaccine may be prophylactic or therapeutic.

Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita, V. et al. (eds.), 1997, Cancer: Principles and Practice of Oncology. Fifth Edition). In one of these strategies, a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 3539-43).

The combinations disclosed herein, e.g., GITR agonist antibody molecules, can be used in conjunction with a collection of recombinant proteins and/or peptides expressed in a tumor in order to generate an immune response to these proteins. These proteins are normally viewed by the immune system as self antigens and are therefore tolerant to them. The tumor antigen may also include the protein telomerase, which is required for the synthesis of telomeres of chromosomes and which is expressed in more than 85% of human cancers and in only a limited number of somatic tissues (Kim, N et al. (1994) Science 266: 2011-2013). (These somatic tissues may be protected from immune attack by various means). Tumor antigen may also be “neo-antigens” expressed in cancer cells because of somatic mutations that alter protein sequence or create fusion proteins between two unrelated sequences (ie. bcr-abl in the Philadelphia chromosome), or idiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated in human cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses (HBV and HCV), Kaposi's Herpes Sarcoma Virus (KHSV), and Epstein-Barr virus (EBV). Another form of tumor specific antigen which may be used in conjunction with a GITR agonist is purified heat shock proteins (HSP) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins from the tumor cells and these HSPs are highly efficient at delivery to antigen presenting cells for eliciting tumor immunity (Suot, R & Srivastava, P (1995) Science 269:1585-1588; Tamura, Y. et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can be used to prime antigen-specific responses. DC's can be produced ex vivo and loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332). DCs may also be transduced by genetic means to express these tumor antigens as well. DCs have also been fused directly to tumor cells for the purposes of immunization (Kugler, A. et al. (2000) Nature Medicine 6:332-336). As a method of vaccination, DC immunization may be effectively combined with other agent, e.g., a GITR agonist, to activate more potent anti-tumor responses.

Bispecific T-Cell Engagers

In one embodiment, a combination described herein includes a bispecific T-cell engager. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a gastrointestinal cancer, a melanoma, or a lung cancer) or a hematologic malignancy (e.g., a lymphoma (e.g., non-Hodgkin's lymphoma) or a leukemia (e.g., an acute lymphoblastic leukemia).

Bi-specific T-cell engagers (BITE®) are a class of artificial bispecific monoclonal antibodies that can direct a host's immune system, e.g., the T cells' cytotoxic activity, against cancer cells. Bi-specific T-cell engagers can form a link between T cells and tumor cells, which causes T cells to exert cytotoxic activity on tumor cells by producing proteins like perforin and granzymes, independently of the presence of MHC I or co-stimulatory molecules. These proteins enter tumor cells and initiate the cell's apoptosis. This action mimics physiological processes observed during T cell attacks against tumor cells.

In some embodiments, the bi-specific T-cell engager is a fusion protein comprising two single-chain variable fragments (scFvs) of different antibodies. In some embodiments, one of the scFvs binds to T cells, e.g., via the CD3 receptor, and the other to a tumor cell, e.g., via a tumor specific molecule.

In some embodiments, the bi-specific T-cell engager is a bispecific antibody molecule of NKG2A and CD138, or a bispecific antibody molecule of CD3 and TCR. In some embodiments, the bispecific T-cell engager is a bispecific antibody molecule that binds to CD3 and a tumor antigen (e.g., EGFR, PSCA, PSMA, EpCAM, HER2 among others).

In some embodiments, the bi-specific T-cell engager is blinatumomab (CAS Registry Number: 853426-35-4). Blinatumomab is also known as MT103. Blinatumomab specifically targets a CD3 site for T cells and a CD19 site for B cells.

In some embodiments, the bi-specific T-cell engager is MT110. MT110 is a single-chain antibody that targets EpCAM and CD3. MT110 is disclosed, e.g., in Amann et al. J Immunother. 2009; 32(5):452-64.

In some embodiments, the bi-specific T-cell engager targets melanoma-associated chondroitin sulfate proteoglycan (MCSP). In some embodiments, the bi-specific T-cell engager targets CD33. In some embodiments the bi-specific T-cell engager comprises trastuzumab (targeting HER2/neu), cetuximab, or panitumumab (both targeting the EGF receptor), a functional fragment thereof. In some embodiments, the bi-specific T-cell engager targets CD66e and EphA2.

Additional GITR Modulators

In some embodiments, the GITR modulator is an antibody molecule, e.g., an anti-GITR antibody molecule as provided herein. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor or a hematologic malignancy.

In other embodiments, exemplary GITR modulators include, e.g., GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as, a GITR fusion protein described in U.S. Pat. No. 6,111,090, European Patent No.: 0920505B1, U.S. Pat. No. 8,586,023, PCT Publication Nos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibody described, e.g., in U.S. Pat. No. 7,025,962, European Patent No.: 1947183B1, U.S. Pat. No. 7,812,135, U.S. Pat. No. 8,388,967, U.S. Pat. No. 8,591,886, European Patent No.: EP 1866339, PCT Publication No.: WO 2011/028683, U.S. Pat. No. 8,709,424, PCT Publication No.:WO 2013/039954, International Publication No.: WO2013/039954, U.S. Publication No.: US2014/0072566, International Publication NO.: WO2015/026684, PCT Publication No.: WO2005/007190, PCT Publication No.: WO 2007/133822, PCT Publication No.: WO2005/055808, PCT Publication No.: WO 99/40196, PCT Publication No.: WO 2001/03720, PCT Publication No.: WO99/20758, U.S. Pat. No. 6,689,607, PCT Publication No.: WO2006/083289, PCT Publication No.: WO 2005/115451, U.S. Pat. No. 7,618,632, PCT Publication No.: WO 2011/051726, International Publication No.: WO2004060319, and International Publication No.: WO2014012479.

In one embodiment, the GITR agonist is used in combination with a PD-1 inhibitor, e.g., as described in WO2015/026684.

In another embodiment, the GITR agonist is used in combination with a TLR agonist, e.g., as described in WO2004060319, and International Publication No.: WO2014012479.

PD-1 Inhibitors

In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab.

In some embodiments, the anti-PD-1 antibody is Nivolumab. Alternative names for Nivolumab include MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558. In some embodiments, the anti-PD-1 antibody is Nivolumab (CAS Registry Number: 946414-94-4). Nivolumab is a fully human IgG4 monoclonal antibody which specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in U.S. Pat. No. 8,008,449 and WO2006/121168. In one embodiment, the inhibitor of PD-1 is Nivolumab, and having a sequence disclosed therein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).

In some embodiments, the anti-PD-1 antibody is Pembrolizumab. Pembrolizumab (also referred to as Lambrolizumab, MK-3475, MK03475, SCH-900475 or KEYTRUDA®; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369 (2): 134-44, U.S. Pat. No. 8,354,509 and WO2009/114335.

In one embodiment, the inhibitor of PD-1 is Pembrolizumab disclosed in, e.g., U.S. Pat. No. 8,354,509 and WO 2009/114335, and having a sequence disclosed therein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).

In some embodiments, the anti-PD-1 antibody is Pidilizumab. Pidilizumab (CT-011; Cure Tech) is a humanized IgGlk monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009/101611.

Other anti-PD1 antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD1 antibodies disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/or US 20120114649.

In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed in WO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1.

In one embodiment, a combination includes an anti-GITR antibody molecule, e.g., as described herein, and an anti-PD-1 antibody disclosed in, e.g., WO 2015/112900, and having a sequence disclosed therein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).

PD-L1 or PD-L2 Inhibitors

In some embodiments, the PD-L1 inhibitor is an antibody molecule. In some embodiments, the anti-PD-L1 inhibitor is chosen from YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

In some embodiments, the anti-PD-L1 antibody is MSB0010718C. MSB0010718C (also referred to as A09-246-2; Merck Serono) is a monoclonal antibody that binds to PD-L1. Pembrolizumab and other humanized anti-PD-L1 antibodies are disclosed in WO2013/079174, and having a sequence disclosed therein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).

In one embodiment, the PD-L1 inhibitor is YW243.55.S70. The YW243.55.S70 antibody is an anti-PD-L1 described in WO 2010/077634 (heavy and light chain variable region sequences shown in SEQ ID Nos. 20 and 21, respectively), and having a sequence disclosed therein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).

In one embodiment, the PD-L1 inhibitor is MDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007/005874, and having a sequence disclosed therein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).

In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech/Roche). MDPL3280A is a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743 and U.S Publication No.: 20120039906.

In other embodiments, the PD-L2 inhibitor is AMP-224. AMP-224 is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1 (B7-DCIg; Amplimmune; e.g., disclosed in WO2010/027827 and WO2011/066342).

LAG-3 Inhibitors

In one embodiment, a combination described herein includes a LAG-3 inhibitor. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor or a hematologic malignancy.

In some embodiments, the anti-LAG-3 antibody is BMS-986016. BMS-986016 (also referred to as BMS986016; Bristol-Myers Squibb) is a monoclonal antibody that binds to LAG-3. BMS-986016 and other humanized anti-LAG-3 antibodies are disclosed in US 2011/0150892, WO2010/019570, and WO2014/008218. In some embodiments, the anti-LAG-3 antibody is a humanized anti-LAG3 antibody disclosed in WO2015/138920.

TIM-3 Inhibitors

In one embodiment, a combination described herein includes a TIM-3 inhibitor. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor or a hematologic malignancy.

Exemplary anti-TIM-3 antibodies are disclosed in U.S. Pat. No. 8,552,156, WO 2011/155607, EP 2581113 and U.S Publication No.: 2014/044728. In some embodiments the anti-TIM3 is a humanized ABTIM3 mAb disclosed in WO2015/117002.

CTLA-4 Inhibitors

In one embodiment, a combination described herein includes a CTLA-4 inhibitor. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor or a hematologic malignancy.

Exemplary anti-CTLA4 antibodies include Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206); and Ipilimumab (CTLA-4 antibody, also known as MDX-010, CAS No. 477202-00-9).

In one embodiment, the combination includes an anti-GITR antibody molecule, e.g., as described herein, and an anti-CTLA-4 antibody, e.g., ipilimumab. Exemplary doses that can be use include a dose of anti-GITR antibody molecule of about 1 to 10 mg/kg, e.g., 3 mg/kg, and a dose of an anti-CTLA-4 antibody, e.g., ipilimumab, of about 3 mg/kg.

Other exemplary anti-CTLA-4 antibodies are disclosed, e.g., in U.S. Pat. No. 5,811,097.

IAP Inhibitors

In one embodiment, a combination described herein includes an inhibitor of Inhibitor of Apoptosis Protein (IAP). In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a breast cancer, an ovarian cancer, or a pancreatic cancer), e.g., a hematologic malignancy (e.g., a multiple myeloma).

In some embodiments, the IAP inhibitor is (S)—N—((S)-1-cyclohexyl-2-((S)-2-(4-(4-fluorobenzoyl)thiazol-2-yl)pyrrolidin-1-yl)-2-oxoethyl)-2-(methylamino)propanamide (Compound A21) or a compound disclosed in U.S. Pat. No. 8,552,003.

In some embodiments, the IAP inhibitor, e.g., (S)—N—((S)-1-cyclohexyl-2-((S)-2-(4-(4-fluorobenzoyl)thiazol-2-yl)pyrrolidin-1-yl)-2-oxoethyl)-2-(methylamino)propanamide (Compound A21) or a compound disclosed in U.S. Pat. No. 8,552,003, is administered at a dose of approximately 1800 mg, e.g., once weekly.

EGFR Inhibitors

In one embodiment, a combination described herein includes an inhibitor of Epidermal Growth Factor Receptor (EGFR). In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a lung cancer (e.g., a non-small cell lung cancer), a pancreatic cancer, a breast cancer, or a colon cancer).

In some embodiments, the EGFR inhibitor is (R,E)-N-(7-chloro-1-(1-(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (Compound A40) or a compound disclosed in PCT Publication No. WO 2013/184757.

In some embodiments, the EGFR inhibitor, e.g., (R,E)-N-(7-chloro-1-(1-(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (Compound A40) or a compound disclosed in PCT Publication No. WO 2013/184757, is administered at a dose of 150-250 mg, e.g., per day. In some embodiments, the EGFR inhibitor, e.g., (R,E)-N-(7-chloro-1-(1-(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (Compound A40) or a compound disclosed in PCT Publication No. WO 2013/184757, is administered at a dose of about 150, 200, or 250 mg, or about 150-200 or 200-250 mg.

In some embodiments, the EGFR inhibitor is chosen from one of more of erlotinib, gefitinib, cetuximab, panitumumab, necitumumab, PF-00299804, nimotuzumab, or RO5083945.

mTOR Inhibitors

In one embodiment, a combination described herein includes an inhibitor of target of rapamycin (mTOR). In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a prostate cancer, a breast cancer, a brain cancer, a bladder cancer, a pancreatic cancer, a renal cancer, or a liver cancer, a lung cancer (e.g., a small cell lung cancer or a non-small cell lung cancer), a respiratory/thoracic cancer, a sarcoma, a bone cancer, a non-small cell lung cancer, an endocrine cancer, an astrocytoma, a cervical cancer, a neurologic cancer, a gastric cancer, or a melanoma), e.g., a hematologic malignancy (e.g., a leukemia (e.g., lymphocytic leukemia), e.g., a lymphoma, or e.g., a multiple myeloma).

In some embodiments, the mTOR inhibitor is dactolisib (Compound A4) or 8-(6-Methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (Compound A41), or a compound disclosed in PCT Publication No. WO 2006/122806.

In some embodiments, the mTOR inhibitor is everolimus (also known as AFINITOR®; Compound A36) or a compound disclosed in PCT Publication No. WO 2014/085318.

In some embodiments, the mTOR inhibitor, e.g., everolimus (Compound A36) or a compound disclosed in PCT Publication No. WO 2014/085318, is administered at a dose of about 2.5-20 mg/day. In one embodiment, the TOR inhibitor, e.g., everolimus (Compound A36) or a compound disclosed in PCT Publication No. WO 2014/085318, is administered at a dose of about 2.5, 5, 10, or 20 mg/day, e.g., about 2.5-5, 5-10, or 10-20 mg/day.

In some embodiments, the mTOR inhibitor is chosen from one or more of rapamycin, temsirolimus (TORISEL®), AZD8055, BEZ235, BGT226, XL765, PF-4691502, GDC0980, SF1126, OSI-027, GSK1059615, KU-0063794, WYE-354, Palomid 529 (P529), PF-04691502, or PKI-587. ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23, 29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); everolimus (AFINITOR® or RAD001); rapamycin (AY22989, SIROLIMUS®); simapimod (CAS Registry Number: 164301-51-3); (5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS Registry Number: 1013101-36-4); N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine inner salt (SF1126, CAS Registry Number: 936487-67-1), or XL765 (SAR245409).

Other exemplary mTOR Inhibitors include, but are not limited to, temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); simapimod; (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502); and N²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-(SEQ ID NO: 378), inner salt (SF1126); and XL765.

IL-15 Agonists

In one embodiment, a combination described herein includes an interleukin-15 (IL-15) agonist. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a refractory solid tumor), (e.g., a melanoma (e.g., a metastatic or advanced melanoma), a kidney cancer (e.g., a renal cell cancer), a non-small cell lung cancer, a squamous cell head and neck cancer, or a bladder cancer (e.g., a non-muscle invasive bladder cancer)), e.g., a hematologic malignancy (e.g., a leukemia, e.g., an acute myelogenous leukemia (e.g., a refractory or relapsed acute myelogenous leukemia), e.g., a lymphoma, e.g., a non-Hodgkin lymphoma (e.g., a relapsed/refractory indolent B cell non-Hodgkin lymphoma), e.g., or a multiple myeloma (e.g., a relapsed or refractory multiple myeloma)).

IL-15, secreted by mononuclear phagocytes (and some other cell types) following viral infection, regulates T and natural killer cell activation and proliferation. This cytokine induces activation of transcription activators STAT3, STATS, and STATE via JAK kinase signal transduction pathways in mast cells, T cells, and dendritic epidermal T cells. IL-15 and interleukin-2 (IL-2) are structurally similar and share many biological activities; both may bind to common hematopoietin receptor subunits, negatively regulating each other's activity. CD8+ memory T cell number can be regulated by a balance between IL-15 and IL-2.

In some embodiments, the IL-15 agonist is a recombinant human IL-15 (rhIL-15), e.g., CYP0150 (Cytune). CYP0150 is a recombinant protein consisting of a human IL-15 linked to the Sushi+ domain of the human alpha chain receptor (transpresentation).

CYP0150 is disclosed, e.g., in PCT Publication No. WO 2007/046006. CYP0150 has the amino acid disclosed as SEQ ID NO: 60 in WO 2007/046006 or disclosed as SEQ ID NO: 62 in WO 2007/046006.

In some embodiments, the IL-15 agonist is ALT-803 (Altor BioScience). ALT-803 is an IL-15N72D:IL-15RαSu/Fc soluble complex, produced from a high-yield recombinant mammalian cell line that co-expresses IL-15N72D and IL-15RαSu/Fc fusion protein. The IL-15 mutant (N72D) has enhanced IL-15 biological activity (Zhu et al. 2009, J Immunol. 183:3598). The IL-15N72D mutant and the soluble domain of IL-15Rα can form stable heterodimeric complexes in solution and this complex exhibits increased biological activity (approximately 25-fold more active) compared to the non-complexed IL-15. ALT-803 is disclosed, e.g., in PCT Publication No. WO 2012/040323 and U.S. Pat. No. 8,507,222.

In some embodiments, the IL-15 agonist is hetlL-15 (Admune). HetIL-15 is a heterodimeric human IL-15 (IL-15/sIL-15Ra). HetIL-15 is disclosed, e.g., in PCT Publication Nos. WO 2009/002562 and WO 2014/066527.

CD40 Agonists

In one embodiment, the combination includes a CD40 agonist. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a lung cancer, an esophageal carcinoma, a melanoma, or a renal cell carcinoma), e.g., a hematologic malignancy (e.g., a leukemia (e.g., a chronic lymphocytic leukemia (CLL)), e.g., a lymphoma (e.g., a non-Hodgkin's lymphoma), e.g., or a multiple myeloma).

In one embodiment, the CD40 agonist is ADC-1013 (Alligator/Biolnvent). ADC-1013 is a fully human IgG agonistic monoclonal antibody against human CD40. CD40, an integral membrane protein found on the surface of B lymphocytes, is a member of the tumor necrosis factor receptor superfamily and is highly expressed in a number of cancers such as B-cell malignancies. CD40 agonists, e.g., anti-CD40 antibodies, are able to substitute effectively for T cell helper activity (Ridge, J. et al. (1998) Nature 393: 474-478).

ADC-1013 is disclosed, e.g., in PCT Publication No. WO 2015/091853. ADC-1013 clones include, e.g., 1136/1137, 1132/1133, 1148/1149, 1140/1135, 1134/1135, 1107/1108, 1142/1135, 1146/1147, and 1150/1151.

The heavy chain variable region of 1132/1133 has the amino acid sequence disclosed as SEQ ID NO: 65 in WO 2015/091853. The light chain variable region of 1132/1133 has the amino acid sequence disclosed as SEQ ID NO: 66 in WO 2015/091853. The heavy chain CDR1 of 1132/1133 has the amino acid sequence disclosed as SEQ ID NO: 13 in WO 2015/091853. The heavy chain CDR2 of 1132/1133 has the amino acid sequence disclosed as SEQ ID NO: 14 in WO 2015/091853. The heavy chain CDR3 of 1132/1133 has the amino acid sequence disclosed as SEQ ID NO: 15 in WO 2015/091853. The light chain CDR1 of 1132/1133 has the amino acid sequence disclosed as SEQ ID NO: 16 in WO 2015/091853. The light chain CDR2 of 1132/1133 has the amino acid sequence disclosed as SEQ ID NO: 17 in WO 2015/091853). The light chain CDR3 of 1132/1133 has the amino acid sequence disclosed as SEQ ID NO: 18 in WO 2015/091853.

The heavy chain variable region of 1107/1108 has the amino acid sequence disclosed as SEQ ID NO: 79 in WO 2015/091853. The light chain variable region of 1107/1108 has the amino acid sequence disclosed as SEQ ID NO: 80 in WO 2015/091853. The heavy chain CDR1 of 1107/1108 has the amino acid sequence disclosed as SEQ ID NO: 55 in WO 2015/091853. The heavy chain CDR2 of 1107/1108 has the amino acid sequence disclosed as SEQ ID NO: 56 in WO 2015/091853. The heavy chain CDR3 of 1107/1108 has the amino acid sequence disclosed as SEQ ID NO: 57 in WO 2015/091853. The light chain CDR1 of 1107/1108 has the amino acid sequence disclosed as SEQ ID NO: 58 in WO 2015/091853. The light chain CDR2 of 1107/1108 has the amino acid sequence disclosed as SEQ ID NO: 59 in WO 2015/091853. The light chain CDR3 of 1107/1108 has the amino acid sequence disclosed as SEQ ID NO: 60 in WO 2015/091853.

In some embodiments, the CD40 agonist is ISF35. ISF35 is a chimeric CD154. ISF is disclosed in PCT Publication Nos. WO 2003/099340 and WO 2008/070743.

In some embodiments, the CD40 agonist is dacetuzumab. Dacetuzumab is also known as SGN-40 or huS2C6. Dacetuzumab is a humanized monoclonal antibody that targets CD40. Dacetuzumab is disclosed, e.g., in Advani et al. J Clin Oncol. 2009; 27(26):4371-7; and Khubchandani et al. Curr Opin Investig Drugs. 2009; 10(6):579-87.

In some embodiments, the CD40 agonist is lucatumumab (CAS Registry Number: 903512-50-5). Lucatumumab is also known as CHIR-12.12 or HCD-122. Lucatumumab binds to and inhibits CD40, thereby inhibiting CD40 ligand-induced cell proliferation and triggering cell lysis via antibody-dependent cellular cytotoxicity (ADCC) in cells overexpressing CD40. Lucatumumab is disclosed, e.g., in Tai et al. Cancer Res. 2005; 65(13):5898-906.

Anti-CD40 antibodies are able to substitute effectively for T cell helper activity (Ridge, J. et al. (1998) Nature 393: 474-478) and can be used in conjunction with PD-1 antibodies (Ito, N. et al. (2000) Immunobiology 201 (5) 527-40).

OX40 Agonists

In one embodiment, a combination described herein includes an OX40 agonist. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a breast cancer, a melanoma, a head and neck cancer, or a prostate cancer), e.g., a hematologic malignancy (e.g., a lymphoma (e.g., a B-cell lymphoma)).

OX40, also known as CD134, is a cell surface glycoprotein and member of the tumor necrosis factor (TNF) receptor superfamily, is expressed on T-lymphocytes and provides a co-stimulatory signal for the proliferation and survival of activated T-cells. OX40 activation can induce proliferation of effector T-lymphocytes, which promotes an immune response against the tumor cells that express tumor-associated antigens (TAAs).

In some embodiments, the OX40 agonist is chosen from mAb 106-222, humanized 106-222 (Hu106), mAb 119-122, or humanized 119-122 (Hu119).

MAb 106-222, humanized 106-222 (Hu106), mAb 119-122, and humanized 119-122 (Hu119) are disclosed, e.g., in PCT Publication No. WO 2012/027328 and U.S. Pat. No. 9,006,399. The amino acid sequence of the heavy chain variable region of mAb 106-222 is disclosed as SEQ ID NO: 4 in WO 2012/027328. The amino acid sequence of the light chain variable region of mAb 106-222 is disclosed as SEQ ID NO: 10 in WO 2012/027328. The amino acid sequence of the heavy chain variable region of humanized 106-222 (Hu106) is disclosed as SEQ ID NO: 5 in WO 2012/027328. The amino acid sequence of the light chain variable region of humanized 106-222 (Hu106) is disclosed as SEQ ID NO: 11 in WO 2012/027328. The amino acid sequence of the heavy chain variable region of mAb 119-122 is disclosed as SEQ ID NO: 16 in WO 2012/027328. The amino acid sequence of the light chain variable region of mAb 119-122 is disclosed as SEQ ID NO: 22 in WO 2012/027328. The amino acid sequence of the heavy chain variable region of humanized 119-122 (Hu119) is disclosed as SEQ ID NO: 17 in WO 2012/027328. The amino acid sequence of the light chain variable region of humanized 119-122 (Hu119) is disclosed as SEQ ID NO: 23 in WO 2012/027328.

In some embodiments, the OX40 agonist is a humanized monoclonal antibody disclosed in U.S. Pat. No. 7,959,925 and PCT Publication No. WO 2006/121810.

In some embodiments, the OX40 agonist is chosen from MEDI6469, MEDI0562, or MEDI6383. MEDI6469 is a murine monoclonal antibody against OX40. MEDI0562 is a humanized monoclonal antibody against OX40. MEDI6383 is a monoclonal antibody against OX40.

In some embodiments, the OX40 agonist, e.g., MEDI6469, is administered intravenously at a dose of approximately 0.4 mg/kg, e.g., every other day.

Other exemplary anti-OX-40 antibodies are disclosed, e.g., in Weinberg, A. et al. (2000) Immunol 164: 2160-2169).

CD27 Agonists

In one embodiment, a combination described herein includes a CD27 agonist. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a melanoma, a renal cell carcinoma, a hormone-refractory prostate adenocarcinoma, an ovarian cancer, a breast cancer, a colorectal adenocarcinoma, or a non-small cell lung cancer), e.g., a hematologic malignancy (e.g., a lymphoma (e.g., a Hodgkin's lymphoma, a Burkett's lymphoma, a mantle cell lymphoma, a primary lymphoma of the central nervous system, or a marginal zone B-cell lymphoma), or a leukemia (e.g., a chronic lymphocytic leukemia (CLL)).

In one embodiment, the CD27 agonist is Varlilumab (CAS Registry Number: 1393344-72-3). Varlilumab is also known as CDX-1127 (Celldex) or 1F5. Varlilumab is a fully human monoclonal antibody (mAb) that targets CD27, molecule in the activation pathway of lymphocytes. CDX-1127 is an agonist anti-CD27 mAb that can activate human T cells in the context of T cell receptor stimulation and therefore mediate anti-tumor effects. CDX-1127 can also provide direct therapeutic effects against tumors with CD27 expression.

Varlilumab is disclosed, e.g., in Vitale et al., Clin Cancer Res. 2012; 18(14):3812-21, WO 2008/051424, and U.S. Pat. No. 8,481,029.

In one embodiment, the CD27 agonist is BION-1402 (BioNovion). BION-1402 is also known as hCD27.15. BION-1402 is an anti-human CD27 monoclonal antibody. BION-1402 can stimulate the proliferation and/or survival of CD27+ cells. BION-1402 can activate human CD27 more effectively than its ligand CD70, which results in a significantly increased effect on proliferation of CD8+ and CD4+ T-cells.

BION-1402 is disclosed, e.g., as hCD27.15 in WO 2012/004367. This antibody is produced by hybridoma hCD27.15, which was deposited with the ATCC in on Jun. 2, 2010 under number PTA-11008. The heavy chain variable region of hCD27.15 has the amino acid sequence disclosed as SEQ ID NO: 3 in WO 2012/004367. The light chain variable region of hCD27.15 has the amino acid sequence disclosed as SEQ ID NO: 4 in WO 2012/004367. The heavy chain CDR1 of hCD27.15 has the amino acid sequence disclosed as SEQ ID NO: 5 in WO 2012/004367. The heavy chain CDR2 of hCD27.15 has the amino acid sequence disclosed as SEQ ID NO: 6 in WO 2012/004367. The heavy chain CDR3 of hCD27.15 has the amino acid sequence disclosed as SEQ ID NO: 7 in WO 2012/004367. The light chain CDR1 of hCD27.15 has the amino acid sequence disclosed as SEQ ID NO: 8 in WO 2012/004367. The light chain CDR2 of hCD27.15 has the amino acid sequence disclosed as SEQ ID NO: 9 in WO 2012/004367. The light chain CDR3 of hCD27.15 has the amino acid sequence disclosed as SEQ ID NO: 10 in WO 2012/004367.

CSF-1/1R Binding Agents

In one embodiment, a combination described herein includes a CSF-1/1R binding agent. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a prostate cancer, a breast cancer, or pigmented villonodular synovitis (PVNS)).

In some embodiments, the CSF-1/1R binding agent is an inhibitor of macrophage colony-stimulating factor (M-CSF).

In another embodiment, the CSF-1/1R binding agent is a CSF-1R tyrosine kinase inhibitor, 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide (Compound A15), or a compound disclosed in PCT Publication No. WO 2005/073224.

In some embodiments, the CSF-1/1R binding agent is an M-CSF inhibitor, Compound A33, or a compound disclosed in PCT Publication No. WO 2004/045532 (e.g., an antibody molecule or Fab fragment against M-CSF).

In some embodiments, the CSF-1/1R binding agent, e.g., an M-CSF inhibitor, Compound A33, or a binding agent to CSF-1 disclosed in PCT Publication No. WO 2004/045532 or PCT Publication No. WO 2005/068503 including RX1 or 5H4 (e.g., an antibody molecule or Fab fragment against M-CSF), is administered at an average dose of about 10 mg/kg. In some embodiments, the CSF-1/1R binding agent is a CSF1R inhibitor or 4-(2-((1R,2R)-2-hydroxycyclohexylamino)benzothiazol-6-yloxy)-N-methylpicolinamide. 4-(2-((1R,2R)-2-hydroxycyclohexylamino)benzothiazol-6-yloxy)-N-methylpicolinamide is disclosed as example 157 at page 117 of PCT Publication No. WO 2007/121484.

In some embodiments, the CSF-1/1R binding agent is pexidartinib (CAS Registry Number 1029044-16-3). Pexidrtinib is also known as PLX3397 or 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-((6-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine. Pexidartinib is a small-molecule receptor tyrosine kinase (RTK) inhibitor of KIT, CSF1R and FLT3. FLT3, CSF1R and FLT3 are overexpressed or mutated in many cancer cell types and play major roles in tumor cell proliferation and metastasis. PLX3397 can bind to and inhibit phosphorylation of stem cell factor receptor (KIT), colony-stimulating factor-1 receptor (CSF1R) and FMS-like tyrosine kinase 3 (FLT3), which may result in the inhibition of tumor cell proliferation and down-modulation of macrophages, osteoclasts and mast cells involved in the osteolytic metastatic disease. In some embodiments, the CSF-1/1R binding agent, e.g., pexidartinib, is used in combination with a GITR agonist, e.g., an anti-GITR antibody molecule provided herein.

In some embodiments, the CSF-1/1R binding agent is emactuzumab. Emactuzumab is also known as RG7155 or RO5509554. Emactuzumab is a humanized IgG1 mAb targeting CSF1R. In some embodiments, the CSF-1/1R binding agent, e.g., pexidartinib, is used in combination with a PD-L1 inhibitor, e.g., an anti-PD-L1 antibody molecule described herein. In some embodiments, the CSF-1/1R binding agent is FPA008. FPA008 is a humanized mAb that inhibits CSF1R. In some embodiments, the CSF-1/1R binding agent, e.g., FPA008, is used in combination with a GITR agonist, e.g., an anti-GITR antibody molecule described herein.

IL-17 Inhibitors

In one embodiment, a combination described herein includes an interleukine-17 (IL-17) inhibitor. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor, e.g., breast cancer, lung cancer, or colon cancer.

In some embodiments, the IL-17 inhibitor is secukinumab (CAS Registry Numbers: 875356-43-7 (heavy chain) and 875356-44-8 (light chain)). Secukinumab is also known as AIN457 and COSENTYX®. Secukinumab is a recombinant human monoclonal IgG1/κ antibody that binds specifically to IL-17A. It is expressed in a recombinant Chinese Hamster Ovary (CHO) cell line.

Secukinumab is described, e.g., in WO 2006/013107, U.S. Pat. No. 7,807,155, U.S. Pat. No. 8,119,131, U.S. Pat. No. 8,617,552, and EP 1776142. The heavy chain variable region of secukinumab has the amino acid sequence disclosed as SEQ ID NO: 8 in WO 2006/013107. The light chain variable region of secukinumab has the amino acid sequence disclosed as SEQ ID NO: 10 in WO 2006/013107. The heavy chain CDR1 of secukinumab has the amino acid sequence disclosed as SEQ ID NO: 1 in WO 2006/013107. The heavy chain CDR2 of secukinumab has the amino acid sequence disclosed as SEQ ID NO: 2 in WO 2006/013107. The heavy chain CDR3 of secukinumab has the amino acid sequence disclosed as SEQ ID NO: 3 in WO 2006/013107. The light chain CDR1 of secukinumab has the amino acid sequence disclosed as SEQ ID NO: 4 in WO 2006/013107. The light chain CDR2 of secukinumab has the amino acid sequence disclosed as SEQ ID NO: 5 in WO 2006/013107. The light chain CDR3 of secukinumab has the amino acid sequence disclosed as SEQ ID NO: 6 in WO 2006/013107.

In some embodiments, the IL-17 inhibitor is CJM112. CJM112 is also known as XAB4. CJM112 is a fully human monoclonal antibody that targets IL-17A.

CJM112 is disclosed, e.g., in WO 2014/122613. The heavy chain of CJM112 has the amino acid sequence disclosed as SEQ ID NO: 14 in WO 2014/122613. The light chain of CJM112 has the amino acid sequence disclosed as SEQ ID NO: 44 in WO 2014/122613.

In some embodiments, the IL-17 inhibitor is ixekizumab (CAS Registry Number: 1143503-69-8). Ixekizumab is also known as LY2439821. Ixekizumab is a humanized IgG4 monoclonal antibody that targets IL-17A.

Ixekizumab is described, e.g., in WO 2007/070750, U.S. Pat. No. 7,838,638, and U.S. Pat. No. 8,110,191. The heavy chain variable region of ixekizumab has the amino acid sequence disclosed as SEQ ID NO: 118 in WO 2007/070750. The light chain variable region of ixekizumab has the amino acid sequence disclosed as SEQ ID NO: 241 in WO 2007/070750.

In some embodiments, the IL-17 inhibitor is brodalumab (CAS Registry Number: 1174395-19-7). Brodalumab is also known as AMG 827 or AM-14. Brodalumab binds to the interleukin-17 receptor A (IL-17RA) and prevents IL-17 from activating the receptor.

Brodalumab is disclosed, e.g., in WO 2008/054603, U.S. Pat. No. 7,767,206, U.S. Pat. No. 7,786,284, U.S. Pat. No. 7,833,527, U.S. Pat. No. 7,939,070, U.S. Pat. No. 8,435,518, U.S. Pat. No. 8,545,842, U.S. Pat. No. 8,790,648, and U.S. Pat. No. 9,073,999. The heavy chain CDR1 of brodalumab has the amino acid sequence disclosed as SEQ ID NO: 146 in WO 2008/054603. The heavy chain CDR2 of brodalumab has the amino acid sequence disclosed as SEQ ID NO: 147 in WO 2008/054603. The heavy chain CDR3 of brodalumab has the amino acid sequence disclosed as SEQ ID NO: 148 in WO 2008/054603. The light chain CDR1 of brodalumab has the amino acid sequence disclosed as SEQ ID NO: 224 in WO 2008/054603. The heavy chain CDR2 of brodalumab has the amino acid sequence disclosed as SEQ ID NO: 225 in WO 2008/054603. The heavy chain CDR3 of brodalumab has the amino acid sequence disclosed as SEQ ID NO: 226 in WO 2008/054603.

IL-1β Inhibitors

In one embodiment, a combination described herein includes an interleukine-1 beta (IL-1β) inhibitor. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a hematologic malignancy (e.g., a lymphoma (e.g., Hodgkin lymphoma), a leukemia (e.g., an acute or chronic leukemia), or a multiple myeloma).

In some embodiments, the IL-1β inhibitor is canakinumab. Canakinumab is also known as ACZ885 or ILARIS®. Canakinumab is a human monoclonal IgG1/κ antibody that neutralizes the bioactivity of human IL-1β.

Canakinumab is disclosed, e.g., in WO 2002/16436, U.S. Pat. No. 7,446,175, and EP 1313769. The heavy chain variable region of canakinumab has the amino acid sequence disclosed as SEQ ID NO: 1 in U.S. Pat. No. 7,446,175. The light chain variable region of canakinumab has the amino acid sequence disclosed as SEQ ID NO: 2 in U.S. Pat. No. 7,446,175.

CXCR2 Inhibitors

In one embodiment, a combination described herein includes an inhibitor of chemokine (C—X—C motif) receptor 2 (CXCR2) inhibitor. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor, e.g., a breast cancer, a metastatic sarcoma, a pancreatic cancer, a melanoma, a renal cell carcinoma (RCC), a non-small cell lung cancer (NSCLC), or a pediatric tumor (e.g., a rhabdomyosarcoma).

In some embodiments, the CXCR2 inhibitor is danirixin (CAS Registry Number: 954126-98-8). Danirixin is also known as GSK1325756 or 1-(4-chloro-2-hydroxy-3-piperidin-3-ylsulfonylphenyl)-3-(3-fluoro-2-methylphenyl)urea. Danirixin is disclosed, e.g., in Miller et al. Eur J Drug Metab Pharmacokinet (2014) 39:173-181; and Miller et al. BMC Pharmacology and Toxicology (2015), 16:18.

In some embodiments, the CXCR2 inhibitor is reparixin (CAS Registry Number: 266359-83-5). Reparixin is also known as repertaxin or (2R)-2-[4-(2-methylpropyl)phenyl]-N-methylsulfonylpropanamide. Reparixin is a non-competitive allosteric inhibitor of CXCR1/2. Reparixin is disclosed, e.g., in Zarbock et al. British Journal of Pharmacology (2008), 1-8.

In some embodiments, the CXCR2 inhibitor is navarixin. Navarixin is also known as MK-7123, SCH 527123, PS291822, or 2-hydroxy-N,N-dimethyl-3-[[2-[[(1R)-1-(5-methylfuran-2-yl)propyl]amino]-3,4-dioxocyclobuten-1-yl]amino]benzamide. Navarixin is disclosed, e.g., in Ning et al. Mol Cancer Ther. 2012; 11(6):1353-64.

PI3K-γ, -δ Inhibitors

In one embodiment, a combination described herein includes an inhibitor of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), e.g., phosphatidylinositol-4,5-bisphosphate 3-kinase gamma and/or delta (PI3K-γ,δ). In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a prostate cancer, a breast cancer, a brain cancer, a bladder cancer, a pancreatic cancer, a renal cancer, a solid tumor, a liver cancer, a non-small cell lung cancer, an endocrine cancer, an ovarian cancer, a melanoma, a female reproductive system cancer, a digestive/gastrointestinal cancer, a glioblastoma multiforme, a head and neck cancer, or a colon cancer), e.g., a hematologic malignancy (e.g., a leukemia (e.g., a lymphocytic leukemia, e.g., chronic lymphocytic leukemia (CLL) (e.g., relapsed CLL)),e.g., a lymphoma (e.g., non-Hodgkin lymphoma (e.g., relapsed follicular B-cell non-Hodgkin lymphoma (FL) or relapsed small lymphocytic lymphoma (SLL)), or e.g., a multiple myeloma).

In some embodiments, the PI3K inhibitor is an inhibitor of delta and gamma isoforms of PI3K. Exemplary PI3K inhibitors that can be used in combination are described in, e.g., WO 2010/036380, WO 2010/006086, WO 09/114870, WO 05/113556, GSK 2126458, GDC-0980, GDC-0941, Sanofi XL147, XL756, XL147, PF-46915032, BKM 120, CAL-101, CAL 263, SF1126, PX-886, and a dual PI3K inhibitor.

In some embodiments, the PI3K-γ,δ inhibitor is idelalisib (CAS Registry Number: 870281-82-6). Idelalisib is also known as ZYDELIG®, GS-1101, CAL-101, or 5-Fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone. Idelalisib blocks P110δ, the delta isoform of PI3K. Idelalisib is disclosed, e.g., in Wu et al. Journal of Hematology & Oncology (2013) 6: 36.

In some embodiments, the PI3K-γ,δ inhibitor is dactolisib (Compound A4) or 8-(6-Methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (Compound A41), or a compound disclosed in PCT Publication No. WO 2006/122806.

In some embodiments, the PI3K-γ,δ inhibitor is buparlisib (Compound A6) or a compound disclosed in PCT Publication No. WO 2007/084786.

In one embodiment, the PI3K-γ,δ inhibitor, e.g., buparlisib (Compound A6) or a compound disclosed in PCT Publication No. WO 2007/084786, is administered at a dose of about 100 mg (e.g., per day).

Other exemplary PI3K-γ,δ inhibitors that can be used in the combination include, e.g., pictilisib (GDC-0941), LY294002, pilaralisib (XL147), PI-3065, PI-103, VS-5584 (SB2343), CZC24832, duvelisib (IPI-145, INK1197), TG100-115, CAY10505, GSK1059615, PF-04691502, AS-605240, voxtalisib (SAR245409, XL765), IC-87114, omipalisib (GSK2126458, GSK458), TG100713, gedatolisib (PF-05212384, PKI-587), PKI-402, XL147 analogue, PIK-90, PIK-293, PIK-294, 3-Methyladenine (3-MA), AS-252424, AS-604850, or apitolisib (GDC-0980, RG7422).

In some embodiments, the PI3K inhibitor is Compound A8 or a compound described in PCT Publication No. WO2010/029082.

In some embodiments, the PI3K inhibitor is a pan-PI3K inhibitor, (4S,5R)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-4-(hydroxymethyl)-5-methyloxazolidin-2-one (Compound A13) or a compound disclosed in PCT Publication No. WO2013/124826.

Exemplary PI3K-γ, -δ inhibitors include, but are not limited to, duvelisib and idelalisib. Idelalisib (also called GS-1101 or CAL-101; Gilead) is a small molecule that blocks the delta isoform of PI3K. The structure of idelalisib (5-Fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone) is shown below.

Duvelisib (also called IPI-145; Infinity Pharmaceuticals and Abbvie) is a small molecule that blocks PI3K-δ,γ. The structure of duvelisib (8-Chloro-2-phenyl-3-[(1S)-1-(9H-purin-6-ylamino)ethyl]-1(2H)-isoquinolinone) is shown below.

In one embodiment, the inhibitor is a dual phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitor selected from 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF-04691502); N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea (PF-05212384, PKI-587); 2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458); 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one Maleic acid (NVP-BGT226); 3-[4-(4-Morpholinylpyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343); or N-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide (XL765).

BAFF-R Inhibitors

In one embodiment, a combination described herein includes a B-cell-activating factor receptor (BAFF-R) inhibitor. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a hematologic malignancy, e.g., a leukemia (e.g., chronic lymphocytic leukemia (CLL), e.g., relapsed or refractory chronic lymphocytic leukemia).

In one embodiment, the BAFF-R inhibitor is VAY736. VAY736 is a fully human combinatorial antibody library (HuCAL)-derived monoclonal antibody targeting BAFF-R. BAFF-R, also known as tumor necrosis factor receptor superfamily member 13C, is overexpressed in certain tumor cell types and autoimmune diseases. VAY736 has both anti-inflammatory and antineoplastic activities. In cancer cells, BAFF-R plays a key role in B-cell proliferation and survival. VAY736 targets and binds to BAFF-R, which inhibits both BAFF/BAFF-R interaction and BAFF-R-mediated signaling. This may decrease cell growth in tumor cells expressing BAFF-R.

VAY736 is disclosed, e.g., in U.S. Pat. No. 8,106,163. The heavy chain CDR1 of VAY736 has the amino acid sequence disclosed as SEQ ID NO: 3 in U.S. Pat. No. 8,106,163. The heavy chain CDR2 of VAY736 has the amino acid sequence disclosed as SEQ ID NO: 10 in U.S. Pat. No. 8,106,163. The heavy chain CDR3 of VAY736 has the amino acid sequence disclosed as SEQ ID NO: 17 in U.S. Pat. No. 8,106,163. The light chain CDR1 of VAY736 has the amino acid sequence disclosed as SEQ ID NO: 24 in U.S. Pat. No. 8,106,163. The light chain CDR2 of VAY736 has the amino acid sequence disclosed as SEQ ID NO: 31 in U.S. Pat. No. 8,106,163. The light chain CDR3 of VAY736 has the amino acid sequence disclosed as SEQ ID NO: 38 in U.S. Pat. No. 8,106,163. The heavy chain variable region of VAY736 has the amino acid sequence disclosed as SEQ ID NO: 52 in U.S. Pat. No. 8,106,163. The light chain variable region of VAY736 has the amino acid sequence disclosed as SEQ ID NO: 45 in U.S. Pat. No. 8,106,163. The heavy chain of VAY736 has the amino acid sequence disclosed as SEQ ID NO: 75 in U.S. Pat. No. 8,106,163. The light chain variable region of VAY736 has the amino acid sequence disclosed as SEQ ID NO: 71 in U.S. Pat. No. 8,106,163.

MALT-1/BTK Inhibitors

In one embodiment, a combination described herein includes an inhibitor of MALT-1 and/or BTK. In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein.

Exemplary BTK inhibitors include, but are not limited to, ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; or LFM-A13. In one embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-inducible kinase (ITK), e.g., is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; or LFM-A13.

In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765). The structure of ibrutinib (1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one) is shown below.

In other embodiments, the BTK inhibitor is a BTK inhibitor described in International Application WO/2015/079417, which is herein incorporated by reference in its entirety. For instance, in some embodiments, the BTK inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof;

wherein,

R1 is hydrogen, C1-C6 alkyl optionally substituted by hydroxy;

R2 is hydrogen or halogen;

R3 is hydrogen or halogen;

R4 is hydrogen;

R5 is hydrogen or halogen;

or R4 and R5 are attached to each other and stand for a bond, —CH2-, —CH2-CH2-, —CH═CH—, —CH═CH—CH2-; —CH2-CH═CH—; or —CH2-CH2-CH2-;

R6 and R7 stand independently from each other for H, C1-C6 alkyl optionally substituted by hydroxyl, C3-C6 cycloalkyl optionally substituted by halogen or hydroxy, or halogen;

R8, R9, R, R′, R10 and R11 independently from each other stand for H, or C1-C6 alkyl optionally substituted by C1-C6 alkoxy; or any two of R8, R9, R, R′, R10 and R11 together with the carbon atom to which they are bound may form a 3-6 membered saturated carbocyclic ring;

R12 is hydrogen or C1-C6 alkyl optionally substituted by halogen or C1-C6 alkoxy;

or R12 and any one of R8, R9, R, R′, R10 or R11 together with the atoms to which they are bound may form a 4, 5, 6 or 7 membered azacyclic ring, which ring may optionally be substituted by halogen, cyano, hydroxyl, C1-C6 alkyl or C1-C6 alkoxy;

n is 0 or 1; and

R13 is C2-C6 alkenyl optionally substituted by C1-C6 alkyl, C1-C6 alkoxy or N,N-di-C1-C6 alkyl amino; C2-C6 alkynyl optionally substituted by C1-C6 alkyl or C1-C6 alkoxy; or C2-C6 alkylenyl oxide optionally substituted by C1-C6 alkyl.

In some embodiments, the BTK inhibitor of Formula I is chosen from: N-(3-(5-((1-Acryloylazetidin-3-yl)oxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (E)-N-(3-(6-Amino-5-((1-(but-2-enoyl)azetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-((1-propioloylazetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-((1-(but-2-ynoyl)azetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(5-((1-Acryloylpiperidin-4-yl)oxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-(2-(N-methylacrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (E)-N-(3-(6-Amino-5-(2-(N-methylbut-2-enamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-(2-(N-methylpropiolamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (E)-N-(3-(6-Amino-5-(2-(4-methoxy-N-methylbut-2-enamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-(2-(N-methylbut-2-ynamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(2-((4-Amino-6-(3-(4-cyclopropyl-2-fluorobenzamido)-5-fluoro-2-methylphenyl)pyrimidin-5-yl)oxy)ethyl)-N-methyloxirane-2-carboxamide; N-(2-((4-Amino-6-(3-(6-cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)phenyl)pyrimidin-5-yl)oxy)ethyl)-N-methylacrylamide; N-(3-(5-(2-Acrylamidoethoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-(2-(N-ethylacrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-(2-(N-(2-fluoroethyl)acrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(5-((1-Acrylamidocyclopropyl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)—N-(3-(5-(2-Acrylamidopropoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)—N-(3-(6-Amino-5-(2-(but-2-ynamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)—N-(3-(6-Amino-5-(2-(N-methylacrylamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)—N-(3-(6-Amino-5-(2-(N-methylbut-2-ynamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-(3-(N-methylacrylamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)—N-(3-(5-((1-Acryloylpyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)—N-(3-(6-Amino-5-((1-(but-2-ynoyl)pyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-2-(3-(5-((1-Acryloylpyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one; N-(2-((4-Amino-6-(3-(6-cyclopropyl-1-oxo-3,4-dihydroisoquinolin-2(1H)-yl)-5-fluoro-2-(hydroxymethyl)phenyl)pyrimidin-5-yl)oxy)ethyl)-N-methylacrylamide; N-(3-(5-(((2S,4R)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-(((2S,4R)-1-(but-2-ynoyl)-4-methoxypyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; 2-(3-(5-(((2S,4R)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one; N-(3-(5-(((2S,4S)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-(((2S,4S)-1-(but-2-ynoyl)-4-methoxypyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(5-(((2S,4R)-1-Acryloyl-4-fluoropyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-Amino-5-(((2S,4R)-1-(but-2-ynoyl)-4-fluoropyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)—N-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)—N-(3-(6-Amino-5-((1-propioloylazetidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-2-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one; (R)—N-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (R)—N-(3-(5-((1-Acryloylpiperidin-3-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(5-(((2R,3S)-1-Acryloyl-3-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(5-(((2S,4R)-1-Acryloyl-4-cyanopyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; or N-(3-(5-(((2S,4S)-1-Acryloyl-4-cyanopyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide.

Unless otherwise provided, the chemical terms used above in describing the BTK inhibitor of Formula I are used according to their meanings as set out in International Application WO/2015/079417, which is herein incorporated by reference in its entirety.

JAK Inhibitors

In one embodiment, a combination described herein includes an inhibitor of Janus kinase (JAK). In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., a colon cancer, a prostate cancer, a lung cancer, a breast cancer, or a pancreatic cancer), e.g., a hematologic malignancy (e.g., a leukemia (e.g., a myeloid leukemia or a lymphocytic leukemia), e.g., a lymphoma (e.g., a non-Hodgkin lymphoma), or e.g., a multiple myeloma.

In some embodiments, the JAK inhibitor is 2-fluoro-N-methyl-4-(7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl)benzamide (Compound A17), or a dihydrochloric salt thereof, or a compound disclosed in PCT Publication No. WO 2007/070514.

In some embodiments, the JAK inhibitor, e.g., 2-fluoro-N-methyl-4-(7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl)benzamide (Compound A17), or a dihydrochloric salt thereof, or a compound disclosed in PCT Publication No. WO 2007/070514, is administered at a dose of about 400-600 mg (e.g., per day), e.g., about 400, 500, or 600 mg, or about 400-500 or 500-600 mg.

In some embodiment, the JAK inhibitor is ruxolitinib phosphate (also known as JAKAFI; Compound A18) or a compound disclosed in PCT Publication No. WO 2007/070514.

In one embodiment, the JAK inhibitor, e.g., ruxolitinib phosphate (also known as JAKAFI; Compound A18) or a compound disclosed in PCT Publication No. WO 2007/070514, is administered at a dose of about 15-25 mg, e.g., twice daily. In some embodiments, the dose is about 15, 20, or 25 mg, or about 15-20 or 20-25 mg.

CRTH2 Inhibitors

In one embodiment, a combination described herein includes an inhibitor of chemoattractant receptor homologous to the T helper 2 cell (CRTH2). In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein.

In some embodiments, the CRTH2 inhibitor is QAV680 (CAS Registry Number: 872365-16-7). QAV680 is also known as fevipiprant and 2-[2-methyl-1-[(4-methylsulfonylphenyl)methyl]pyrrolo[2,3-b]pyridin-3-yl]acetic acid. QAV680 is disclosed, e.g., in Sandham et al. Bioorg Med Chem. 2013; 21(21):6582-91.

In some embodiments, the CRTH2 inhibitor is QAW039 (CAS Number: 872365-14-5). QAW039 is also known as [1-(4-Methanesulfonyl-2-trifluoromethyl-benzyl)-2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl]-acetic acid. QAW039 is disclosed, e.g. in Sykes et al. European Respiratory Journal Sep. 1, 2014 vol. 44 no. Suppl 58 P4074.

Other CRTH2 inhibitors that can be used in the combination include, e.g., AZD1981, ARRY-502, setipiprant (ACT-453859), and ACT-129968.

PFKFB3 inhibitors

In one embodiment, a combination described herein includes an inhibitor of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). In some embodiments, the combination is used to treat a cancer, e.g., a cancer described herein, e.g., a solid tumor (e.g., an advanced solid tumor).

In some embodiments, the PFKFB3 inhibitor is PFK-158. PFK-158 is also known as ACT-PFK-158 or (E)-1-(pyridyn-4-yl)-3-(7-(trifluoromethyl)quinolin-2-yl)-prop-2-en-1-one. PFK-158 is a derivative of 3-(3-pyridinyl)-1-[4-pyridinyl]-2-propen-1-one (3P0). PFKFB3, which catalyzes the conversion of fructose-6-phosphate to fructose-2,6-bisphosphate, is highly expressed and active in human cancer cells and plays a key role in increasing both glycolytic flux in and proliferation of cancer cells. PFKFB3 inhibitors, e.g., PFK-158, can bind to and inhibit the activity of PFKFB3, which leads to the inhibition of both the glycolytic pathway in and glucose uptake by cancer cells. This prevents the production of macromolecules and energy that causes the enhanced cellular proliferation in cancer cells as compared to that of normal, healthy cells. Depriving cancer cells of nutrients and energy leads to the inhibition of cancer cell growth. PFK158 is disclosed, e.g., at page 5 of WO 2013/148228.

In some embodiments, the PFKFB3 inhibitor has the following structure:

In another aspect, the invention features a composition (e.g., one or more compositions or dosage forms), that includes a combination of three or more therapeutic agents chosen from one, two or all of the following categories (i)-(iii): (i) an agent that enhances antigen (e.g., tumor antigen) presentation; (ii) an agent that enhances an effector cell response (e.g., activation and/or mobilization of B cell and/or T cell); or (iii) an agent that decreases tumor immunosuppression. In some embodiments, the combination includes a a-GITR modulator (e.g., an anti-GITR antibody molecule as described herein).

In yet another aspect, the invention features a composition (e.g., one or more compositions or dosage forms as described hereom), for use in treating a disorder, e.g., a cancer. In embodiments, the composition for use includes a combination of two, three or more therapeutic agents chosen from one, two or all of the following categories (i)-(iii): (i) an agent that enhances antigen (e.g., tumor antigen) presentation; (ii) an agent that enhances an effector cell response (e.g., activation and/or mobilization of B cell and/or T cell); or (iii) an agent that decreases tumor immunosuppression. In some embodiments, the combination used includes a a-GITR modulator (e.g., an anti-GITR antibody molecule as described herein). The cancer can be, e.g., a cancer described herein, such as lung cancer (squamous), lung cancer (adenocarcinoma), head and neck cancer, cervical cancer (squamous), stomach cancer, thyroid cancer, melanoma, nasopharyngeal cancer, or breast cancer.

The combinations of therapeutic agents disclosed herein include two or more therapeutic agents described herein. The therapeutic agents in the combination can belong to the same category, e.g., two or more therapeutic agents of category (i), or can include at least one agent of two or more categories (e.g., a therapeutic agent of category (i) combined with a therapeutic agent of category (ii)), as described below. Certain therapeutic agents can belong to two or more categories of categories (i)-(iii). For example, a therapeutic agent (e.g., a GITR agonist, an IDO antagonist, a TGF-b inhibitor, among others) can act as a therapeutic agent in multiple categories.

Formulations, e.g., dosage formulations, and kits, e.g., therapeutic kits, that include a combination of two, three or more therapeutic agents chosen from one, two or all of the following categories (i)-(iii): (i) an agent that enhances antigen (e.g., tumor antigen) presentation; (ii) an agent that enhances an effector cell response (e.g., activation and/or mobilization of B cell and/or T cell); or (iii) an agent that decreases tumor immunosuppression, thereby reducing an activity in the cell, and (optionally) instructions for use, are also disclosed. In some embodiments, the combination includes a a-GITR modulator (e.g., an anti-GITR antibody molecule as described herein).

The combinations disclosed herein can be administered together in a single composition or administered separately in two or more different compositions, e.g., compositions or dosage forms as described herein. The administration of the therapeutic agents can be in any order. The first agent and the additional agents (e.g., second, third agents) can be administered via the same administration route or via different administration routes. For example, a first therapeutic agent can be administered concurrently with, prior to, or subsequent to, the additional agent. In certain embodiments, a first agent is administered locally, e.g., a therapeutic agent of any of categories (i)-(iii) can be coupled to a tumor targeting agent, e.g., a tumor-targeting antibody (e.g., to form an antibody-drug conjugate), or any other delivery agent (e.g., a formulation such as a targeted formulation) such that administration of the first agent is localized to a desired site, e.g., a tumor site (e.g., a dendritic cell-enriched site). In one embodiment, the therapeutic agent is an antigen (e.g., a vaccine, e.g., an in situ cancer vaccine), which is targeted to the tumor environment, thus resulting in activation of dendritic cells. The therapeutic agent also can be locally administered, e.g., injected, at a tumor site (e.g., intratumoral or peritumoral administration). Localized delivery or administration of the therapeutic agent can reduce one or more side effects or toxicities that would otherwise be associated with systemic administration of the therapeutic agent. In one exemplary embodiment, a therapeutic agent (e.g., STING or a TLR) can be conjugated to a tumor-binding antibody (e.g., an antibody that binds a tumor associated antigen (TAA)), thereby delivering the therapeutic agent to a TAA-expressing cell.

In other embodiments, the combination includes a therapeutic agent from the antigen-presentation combination (e.g., one or more of a STING agonist, a TLR agonist, a vaccine or an oncolytic virus) in combination with a an anti-GITR antibody molecule as described herein and a therapeutic agent from the effector cell and/or anti-tumor immunosuppression combination (e.g., an inhibitor of a checkpoint inhibitor, e.g., an inhibitor of PD-1, PD-L1, LAG-3, TIM-3, CEACAM (e.g., CEACAM-1, -3 and/or -5), or CTLA-4, or any combination thereof for therapy. In one embodiment, one or more of a STING agonist, a TLR agonist, a vaccine or an oncolytic virus is administered in combination with an anti-GITR antibody molecule as described herein. In one embodiment, a STING agonist and/or a vaccine is administered in combination with an anti-GITR antibody molecule as described herein. In one embodiment, an oncolytic virus is administered in combination with an anti-GITR antibody molecule as described herein.

When administered in combination, the first agent, the additional agent (e.g., second or third agent), or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of the first agent, the additional agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy. In other embodiments, the amount or dosage of the first agent, the additional agent (e.g., second or third agent), or all, that results in a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower).

Provided combinations can be used to treat a cancer as described herein. Cancers subject to treatment include without limitation epithelial cancers or carcinomas, as well as sarcomas and lymphomas. In some embodiments, the cancer is melanoma, ovarian cancer, renal cancer, colorectal cancer, prostate, lung cancer including non-small cell lung cancer (NSCLC), breast cancer, glioma, or fibrosarcoma. In some embodiments, the type of cancer is selected from the group consisting of: pancreatic cancer, melanomas, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues and head and neck squamous cell carcinoma (HNSCC). One or more combinations can be used to treat a cancer as described herein, such as pancreatic cancer, melanomas, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues and head and neck squamous cell carcinoma (HNSCC). Methods and compositions disclosed herein are useful for treating metastatic lesions associated with the aforementioned cancers.

Still further, the invention provides a method of enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) the antigen; and (ii) a combination as described herein, e.g., a combination comprising a therapeutically effective amount of an anti-GITR antibody molecule described herein, such that an immune response to the antigen in the subject is enhanced. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen or an antigen from a pathogen.

The combinations as described herein can be administered to the subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation or intracavitary installation), topically, or by application to mucous membranes, such as the nose, throat and bronchial tubes.

Dosages and therapeutic regimens of the therapeutic agents disclosed herein can be determined by a skilled artisan. In certain embodiments, the anti-GITR antibody molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about 3 mg/kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks. In one embodiment, the anti-GITR antibody molecule is administered at a dose from about 10 to 20 mg/kg every other week.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D illustrate epitope mapping of the GITR mAbs of the invention. FIG. 1A depicts results of hydrogen/deuterium exchange coupled to mass spectrometry (HDXMS) analyses using Fc-GITR fusion (top and middle trace) and HIS-GITR (lower trace) fusion proteins and MAB1 parental Ab. Numbering reflects removal of native GITR signal peptide (AA 1-26) sequence. FIG. 1B depicts a schematic of N-terminal deletion constructs prepared using the extracellular domain of human GITR (hGITR ECD). FIG. 1C depicts results of binding of MAB4 and MAB5 to hGITR ECD constructs. N-terminal deletion of cysteine-rich domain 1 (CRD1) from human GITR (hGITR) extracellular domain (ECD) abrogates binding of MAB4 and MAB5 to hGITR. Similar results were obtained for MAB7 (data not shown). FIG. 1D depicts results of alanine scanning mutagenesis. MAB7 bound to all mutant proteins with the exception of GITR mutant E78A. ForteBio™ binding analysis was carried out, and results also confirmed loss of MAB7 binding to hGITRE78A mutant protein (data not shown). Results implicates a region of ECD of GITR including CRD1 and including E78 (SEQ ID NO:88: RPTGGPGCGPGRLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGD) as a region and potential epitope involved in binding MAB1 and MAB7 (parental mAb).

FIG. 2A-2E depicts results of binding experiments of anti-GITR MAB antibodies. FIGS. 2A and 2B illustrates MAB4, and MAB5 specifically bind to GITR from human and cynomolgus monkeys (2A) but not from rodent (2B), as determined by ELISA assays. FIG. 2C illustrates MAB7 shares a similar profile binding human and cyno GITR but not murine GITR by ELISA assay. FIG. 2D illustrates MAB7 competes with GITR-ligand binding as determined by FACS competition analysis. FIG. 2E illustrates results of ELISA assays showing that the anti-GITR antibodies of the invention (e.g., MAB4, MAB5) do not bind to other members of the TNF receptor superfamily (TNFRSF). Protagen™ chip assays also confirmed that the antibodies do not bind to other off-target proteins (not shown).

FIG. 3A-3D depict intracellular signaling in 293 cells that have been engineered to express GITR. FIG. 3A illustrates that recombinant human GITR ligand (GITR-L) activates intracellular signaling in 293 cells that have been stably transfected to overexpress human GITR. FIG. 3B illustrates that monoclonal antibodies MAB4 and MAB5 activate intracellular signaling in 293 cells that have been transfected to overexpress human GITR comparably to GITR-L when the antibodies are cross-linked (EC₅₀ for GITRL is about 65 nM versus EC₅₀ of about 2.5 nM for agonist antibodies in the presence of cross-linker). FIG. 3C illustrates that cross-linked MAB antibody activates intracellular signaling in cells, as MAB7 and MAB8 also promote NFκB activation in 293 cells stably transfected with human GITR and the NFkB-Luciferase reporter gene in a similar manner to cross-linked MAB4. FIG. 3D illustrates that Cross-linked MAB4 and MAB5 promote NFκB activation in 293 cells that have been stably transfected with cyno GITR and the NFκB-Luciferase reporter gene. Similar activation was seen with cross-linked MAB7 (data not shown).

FIG. 4A-4C depicts in vitro co-stimulatory activity of MAB7 on T cells is dependent upon T cell activation. Anti-CD3 (OKT3), anti-CD28 (CD28.2) and anti-GITR mAbs were cross-linked (at a ratio of 1:1:3) on beads and then incubated with PBMCs. FIG. 4A illustrates MAB7 is a co-activator of CD4+ T cells and stimulates T cell proliferation in CD4+ Tcells. FIG. 4B illustrates MAB7 is a co-activator of CD8+ T cells and stimulates T cell proliferation in CD8+ Tcells. FIG. 4C illustrates cytokine production, e.g. IFNγ production following TCR engagement is increased in conjunction with MAB7. Similar results were seen for MAB4 and 5 (data not shown).

FIG. 5A-D illustrates in vitro ADCC activity of MAB7 in GITR expressing cells at varying levels. FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D each depict results of ADCC activity using control or MAB7 antibody with various levels of GITR expression. MAB7 is able to induce signaling through the FcgRllla, with increased activity upon increased levels of GITR signaling.

FIG. 6A-6D illustrates GITR is functional in hGITR-hGITRL knock-in mice. Splenocytes were isolated from hGITR-hGITRL knock-in mice and cultured either unstimulated or stimulated with aCD3 and aCD8 antibodies for 48 hours, then pulsed with controls or MAB7 at varying concentrations for 30 minutes, then fixed and stained with fluorophore-conjugated antibodies and analyzed by flow cytometry. FIG. 6A depicts results showing expression of hGITR is upregulated on stimulated CD8+ T cells. FIG. 6B depicts results of anti hGITR antibody binding to T cells by hFc staining, showing MAB7 can bind to hGITR expressed on mouse CD8+ T cells. FIGS. 6C and 6D depict MAB7 binding to stimulated CD8+ T cells correlates with increased T cell activation, as shown by intracellular pIKK staining (6C) and T cell activation (6D). *p<0.05, **p<0.005.

FIG. 7A-7C illustrates MAB7 is functional in vivo. hGITR-hGITRL double knock-in mice with established Colon26 tumors were treated with a single dose of vehicle (n=8/timepoint) or MAB7 (n=10/timepoint) antibody. FIG. 7A depicts results of tumor measurements twice per week and tumor volume calculated using the equation (L×W²)/2. Data shown is from the fifteen (15)-day time point group. FIGS. 7B and 7C depict results from whole blood and FIGS. 7C and 7D depict results from tumors were collected 3-days post-dose and analyzed by flow cytometry for cell surface hGITR expression on immune cells. (*p<0.05, ****p<0.00005).

FIG. 8A-8E illustrates MAB7 elicits an anti-tumor immune response to Colon26 tumors in vivo. hGITR-hGITRL double knock-in mice with established Colon26 tumors were treated with a single dose of vehicle (n=8/timepoint) or MAB7 (n=10/timepoint). FIG. 8A depicts results of Tregulatory cells 3-days post-dose. FIG. 8B-8C depict results of lymphocytes (8B) and activated CD8+ T cells (8C) present in tumor site following treatment levels in tumor 15-days post-dose. The absolute number of cells was normalized to tumor size to account for the significant difference in tumor size between Vehicle and MAB7 treated groups. FIG. 8D depicts Teff/Treg ratio resulting in treated animals as determined by total intratumoral activated CD8+ T cells compared to CD4+ FOXP3+ Tregs to generate T_(eff)/T_(reg) ratios. FIG. 8E depicts results of splenocyte assays from purified CD8+ T cells incubated with Colon26 tumor cells ex-vivo, and measuring CTL response using IFNg ELISPOT assay. (*p<0.05, ***p<0.0005).

FIG. 9A-9C illustrates PD-1 expression is upregulated on CD8+ T cells in Colon26 tumors as well as spleens after treatment with a murine surrogate GITR antibody, DTA-1. Single cell suspensions of whole tumors or whole spleens were profiled by flow cytometry following 2 doses of DTA-1. FIG. 9A depicts results of PD-1 positive cells assessed as a percentage of total CD19-CD3+CD8+ T cells. FIG. 9B depicts results of PD-1 positive cells normalized to tumor size by absolute number of PD-1+CD19-CD3+CD8+ T cells per mm³ volume of tumor. FIG. 9C depicts results of PD-1 expression is upregulated on CD8+ T cells in spleens of Colon26 tumor bearing mice after treatment with DTA-1. PD-1 positive cells were assessed as a percentage of total CD19-CD3+CD8+ T cells. (*p<0.05 and ****p<0.0005).

FIG. 10 illustrates anti-GITR and anti-PD-1 combinations confer survival advantage compared to isotype control. Depicted are results in Colon26 mice models treated with anti-GITR (IgG2a-DTA-1) and anti-PD-1 (RMP1-14) individually or in combination as compared to isotype control.

FIG. 11 illustrates expression of LAG3(first column), TIM3(middle column), and PD1(right column) after treatment with anti-GITR, anti-PD-1 and anti-GITR/anti-PD-1 in combinations in mice with established Colon26 tumors as compared to treatment with isotype control Ab. Depicted are results in Colon26 mice models treated with anti-GITR (IgG2a-DTA-1) and anti-PD-1 (RMP1-14) individually or in combination as compared to isotype control. The top row demonstrates results in tumor samples, and the lower row depicts results in spleen samples. LAG3, TIM3 and PD1 expression is upregulated on CD8+ T cells in Colon26 tumors after treatment with a-GITR, and a-PD1 PD-1 expression is upregulated on CD8+ T cells in Colon26 tumors after treatment with anti-GITR/anti-PD-1 in combination.

EXEMPLIFICATION Creation of GITR Agonist Antibodies MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8

Human antibodies MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 were generated by engineering a murine monoclonal GITR agonist antibody MAB1 to have greater sequence homology to a human germline antibody. MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 retain the epitope specificity, affinity, and cynomolgus macaque GITR cross-reactivity of the parental murine antibody, MAB1. MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 have much higher homology to the human germline sequence than the original murine antibody and should therefore be better tolerated by the human immune system.

Mouse monoclonal MAB1 was engineered to bring its protein sequence closer to a human germline sequence and decrease its immunogenicity using the Humaneered® technology platform available through KaloBios, (South San Francisco, Calif. (on the worldwide web at kalobios.com)). Humaneered® antibodies are very close to human antibodies with V-region sequences that have high homology to a human germline sequence while still retaining the specificity and affinity of the parent or reference antibody (U.S. Patent Publ. 2005/0255552 and 2006/0134098). The process first identifies the minimum antigen binding specificity determinants (BSDs) in heavy and light chain variable regions of a reference Fab (typically sequences within the heavy chain CDR3 and the light chain CDR3). As these heavy and light chain BSDs are maintained in all libraries constructed during the process, each library is epitope-focused, and the resulting Humaneered® antibodies retain the epitope specificity of the original mouse antibody.

Next, full chain libraries (in which an entire light or heavy chain variable region is replaced with a library of human sequences) and/or cassette libraries (in which a portion of the heavy or light chain variable region of the mouse Fab is replaced with a library of human sequences) are generated. A bacterial secretion system is used to express members of the library as antibody Fab fragments, and the library is screened for Fabs that bind antigen using a colony lift binding assay (CLBA). Positive clones are further characterized to identify those with the highest affinity. Identified human cassettes supporting binding in the context of residual murine sequences are the combined in a final library screen to generate completely human V-regions.

Resulting Humaneered® antibody Fabs have V-segment sequences derived from human libraries, retain the short BSD sequences identified within the CDR3 regions, and have human germline Framework 4 regions. These Fabs are converted to full IgGs by cloning variable regions of the heavy and light chains into IgG expression vectors. Humaneered® antibodies generated in this process retain the binding specificity of the parent, murine antibody, typically having equivalent or higher affinity for antigen that the parent antibody, and have V-regions with a high degree of sequence identity compared with human germline antibody genes at the protein level.

Methods Generation of Murine Anti-GITR mAb MAB1

Bcl-2 transgenic mice (C57BL/6-Tgn (bcl-2) 22 wehi strain) were immunized with the N-terminal region of human GITR (aa 26-161) using a procedure that calls for Repetitive Immunization at Multiple Sites (RIMMS) (McIntyre G D. Hybridoma 1997) followed by hybridoma generation from high titer mice. A hybridoma secreting MAB1 was identified and selected using a sandwich ELISA against hGITR and an NFκB Reporter Gene Assay to confirm hGITR binding and agonist activity.

Cloning of Murine V-Regions

Variable region DNA from murine monoclonal MAB1 was amplified by RT-PCR from RNA obtained from the hybridoma cell line using standard methods. Heavy chain variable region was amplified from MAB1 cDNA with HV3 (5′-GGGTCTAGACACCATGGCTGTCTTGGGGCTGCTCTTC-3′ (SEQ ID NO:95)) and HCconstant (5′-GCGTCTAGAAYCTCCACACACAGGRRCCAGTGGATAGAC-3′ (SEQ ID NO:96)). Light chain variable region was amplified from the same cDNA with LV3 (5′-GGGTCTAGACACCATGGAGWCACAKWCTCAGGTCTTTRTA-3′ (SEQ ID NO:97)) and LCconstant (5′-GCGTCTAGAACTGGATGGTGGGAAGATGG-3′ (SEQ ID NO:19)). Variable heavy and light chain products were inserted into a pcDNA3.1 vector and sequence verified. The heavy and light vectors were used as templates for PCR incorporating restriction enzyme sites for cloning into KaloBios vectors: Vh into KB1292-His (modified version of KB1292 that encodes a C-terminal flexible linker and 6-His tag (SEQ ID NO:11) of amino acid sequence AAGASHHHHHH (SEQ ID NO:13) on CH1) at NcoI (5′) and Nhel (3′); Vk into KB1296 at NcoI (5′) and BsiWI (3′). These separate heavy and light chain vectors were then combined into a single dicistronic KaloBios Fab expression vector by restriction digest with BssHII and ClaI and ligation. Fab fragments were expressed in E. coli from this vector. This Fab was tested for hGITR-antigen binding and is referred to as MAB1rFab.

Fab Purification

Fab fragments were expressed by secretion from E. coli using KaloBios expression vectors. Cells were grown in 2×YT medium to an OD500 of ˜0.6. Expression was induced by adding IPTG to 100 μM and shaking for 4 hours at 33° C. Assembled Fab was obtained from periplasmic fractions by osmotic lysis and purification by affinity chromatography using Ni-NTA columns HisTrap HP columns; GE Healthcare catalog #17-5247-01) according to standard methods. Fabs were eluted in buffer containing 500 mM imidazole and thoroughly dialyzed against PBS pH7.4 without calcium and magnesium.

Library Construction

To limit the complexity to identify complimentary human CDRs that support BSD-FR4 in human GITR binding, a cassette library approach, in which only part of the parent murine V-segment is replaced by a library of human sequences, was taken. The original murine MAB1 Vk is closest to human germline VkIII, so a mixture of two KaloBios libraries that contains VkIII germlines (KB1423 and KB1424) was used in making the Vk cassette libraries. KaloBios libraries that contains VH3 germlines (KB1413, KB1414) were used to construct Vh cassette libraries. Two types of cassettes were constructed by overlap PCR: front-end cassettes (8C1VK3FE-01, and MAB1VH3FE-01) containing human sequences in FR1, CDR1, and FR2, and FR3 cassettes (MAB1VK3FR3-01, and MAB1VH3FR3-01) containing human sequences in the FR3 were amplified using the above mentioned germline restricted KaloBios libraries. Each Vh cassette library was cloned into vector KB1292-His at NcoI (5′) and KpnI (3′); each Vk cassette library was cloned into vector KB1296-B (modified version of KaloBios vector KB1296 which has a silent HindIII site added in FR4) at NcoI (5′) and HindIII (3′). Resultant Vh or Vk plasmid libraries were then combined with the complementary chain from the optimized reference Fab (MAB1opVK or MAB1 opVH (e.g., the Vh front-end library was combined with the optimized reference Vk vector) by digestion with BssHII and ClaI and subsequent ligation to create libraries of dicistronic vectors expressing full Fabs.

No VH3 front-end clones bound the human GITR with high affinity, thus, a second VH3 front-end library (MAB1VH3FE-02) was constructed. This library contains human sequences in FR1, FR2, and a collection of CDR2s encoding either the parental murine residue or the selected human germline residue at all positions. The FR3 region sequences of this library were from six clones selected from the VH3FR3 library (MAB1VH3FR3-01).

The final Vk full-chain library (MAB1VK3FCL-01) was constructed by combining clones from VK front-end and VKFR3 cassette libraries with mutagenic VK CDR2s that encodes either the parental murine or the selected human germline residue at all positions. The resulting Vk full-chain library was cloned into KB1296b at NcoI and HindIII sites. This VK full-chain library was paired with a number of selected VH3FR3 library clones to allow functional Fab expression and screened by CLBS. The antigen specific clones were confirmed by human GITR specific ELISA and ranked by antigen affinity titration ELISA. The VH3 full-chain library (MAB1VH3FcL-01) was generated using the selected clones from the second VH3 front end library (MAB1VH3FE-02) with a collection of CDR2 sequences containing either the parental murine or human residue at each position. This VH full-chain library was cloned into KB1292-his at NcoI and KpnI sites. To yield the final full-chain human Fab expression library, selected VK full-chain clones were combined with VH full-chain library at BssHII and ClaI sites.

General ELISA

Recombinant human GITR and human Fc fusion protein (hGITR-hFc) was used for all ELISA assays. Typically, hGITR-hFc antigen diluted in PBS pH 7.4 was bound to a 96-well microtiter plate at 200 ng/well by overnight incubation at 4° C. After being rinsed three times with PBST, the plate was blocked with a solution of 1% BSA in PBS for one hour at 37° C., and then rinsed once with PBST. Fab-containing cell medium or diluted, purified Fab (50 μL) was then added to each well. After a one-hour incubation at 37° C., or overnight incubation at 4° C., the plate was rinsed three times with PBST. Anti-human-kappa chain HRP conjugate (Sigma #A7164) diluted 1:5000 in PBST (50 μL) was added to each well, and the plate was incubated for 45 min at room temperature. The plate was washed three times with PBST, then 100 μL of SureBlue TMB substrate (KPL #52-00-03) was added to each well and the plate was incubated for about 10 min at room temperature. The plate was read at 650 nm in a spectrophotometer.

Affinity Titration ELISA

In order to evaluate antigen binding of the selected Fab producing clones, an affinity titration ELISA was developed. This assay combines two consecutive ELISA steps: the first one, using goat anti-human Fab (Jackson ImmunoResearch Lab #109-005-097) capture and goat anti-human Kappa (Sigma #A7164) detection, measures Fab concentrations in cell culture medium to normalize the amount of Fab used in the second antigen titration ELISA; the second ELISA, a normal antigen specific ELISA, generates an antigen binding dilution curve with the same amount of starting Fab. By comparing the dilution curves of different clones the high affinity clones are identified.

Colony Lift Binding ELISA (CLBA)

Screening of Humaneered® antibody libraries of Fab fragments was carried out essentially as described in (U.S. Patent Publ. 2005/0255552 and 2006/0134098) using nitrocellulose filters coated with hGITR-hFC at 2.0 μg/mL in PBS pH7.4. Fabs bound to antigen-coated filter were detected using goat anti-human Kappa chain HRP conjugate (Sigma #A7164) diluted 1:5000 in PBST, and blots were developed with ECL plus Western Blotting Detection System (GE Healthcare #RPN2132).

Removal of Glycation Site in MAB4

A glycation site “KH” in the junction of FR3 and CDR3 of MAB4 heavy chain was removed by replacing the lysine with an arginine, or replacing the lysine with an arginine and the histidine with an Asparagine. The KH to RH and KH to RN conversions were accomplished by PCR based mutagenesis using the p50H plasmid as the DNA template. The reverse primer (TCTGGCGCAGTAATACACGGCC, SEQ ID NO:110) incorporated an arginine in place of the lysine; the forward primer (NNKGCCTATGGCCATGATGGCG, SEQ ID NO:111) had the degenerate NNK trinucleotide at the histidine site. PCR reactions were performed with 100 ng of template, 0.2 μM of each primer, 200 μM dNTPs, and 2.5 U of pfuUltraII DNA polymerase (Strategene) in a 50 μl reaction volume. The PCR conditions were 94° C. for 3 min for 1 cycle; 94° C. for 15 seconds, 52° C. for 20 seconds, and 65° C. for 5 minutes for 30 cycles; and finally, 1 cycle at 72° C. for 5 minutes. Dpnl (2 U) was added to the PCR reaction and incubated at 37° C. for 30 minutes to remove the template p50H. Amplified MAB4 heavy chain variants were separated by a 1% SYBR gel and purified using a Qiagen Gel Purification Kit. The gel purified PCR product was treated with T4 DNA polynucleotide kinase, ligated and transformed into DH5a chemically competent cells (Invitrogen) under ampicillin selection.

Clones hosting the MAB7 and MAB8 heavy chain were selected by colony PCR using the forward (GCCTTTCTCTCCACAGG, SEQ ID NO:112) and reverse (GGCAAACAACAGATGGCTGG, SEQ ID NO:113) primers following GoTaqClear protocol (Promega). The PCR conditions were 94° C. for 3 minutes for 1 cycle; 94° C. for 10 seconds, 55° C. for 30 seconds, 72° C. for 45 seconds for 25 times; and finally, one cycle at 72° C. for 5 minutes. PCR reactions were cleaned up for sequencing by incubating the samples at 37° C. for 30 minutes and 80° C. for 15 minutes with Exonuclease I and Shrimp Alkaline Phosphotase. PCR samples were sequenced and the results were analyzed using Clone Manager software.

Antibody Production and Purification

Generated antibodies MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 (IgG1 kappa) were produced by co-transfection of vectors as follows into 293 Freestyle cells using 293fectin transfection reagent (Invitrogen #51-0031) according to the manufacturer's protocol.

-   -   MAB2—p35H+p35kappa     -   MAB3—p38H+p38kappa     -   MAB4—p50H+p35kappa     -   MAB5—p51H+p35kappa     -   MAB6—p56H+p35kappa     -   MAB7—pMAB7H+p35kappa     -   MAB8—pMAB8H+p35kappa

Antibody was purified from 293 Freestyle cells supernatants using a 5-mL HiTrap Protein A HP column (GE Healthcare #17-0403-03). Antibody was eluted using IgG Elution Buffer (Pierce #21004), and buffer exchanged into PBS by dialysis. Protein A affinity chromatography was performed on an AKTA-FPLC liquid chromatography system (GE Healthcare).

Specificity ELISA

For the specificity ELISA, a crude cell lysate was made from bacteria expressing members of the TNFRSF family. To prevent nonspecific binding to the plate 50 μL of 5% BSA was added per mL of bacterial lysate. A HisGrab Nickel 96-well plate (Pierce #15142) was coated with TNFRSF containing bacterial lysate at 100 μL of lysate/BSA per well and incubated for 1 hour at room temperature. The plate was then rinsed three times with PBST, then MAB was diluted to 0.5 μg/mL in 10% FBS in PBS and 100 μL was added to each well. The plate was incubated for 1 hour at room temperature and then rinsed three times with PBST. Anti-human kappa antibody (Sigma #A7164) conjugated to HRP was diluted 1:5000 in 1:1 PBST: 10% FBS in PBS and 100 μL added to each well. The plate was incubated for 1 hour at room temperature, and then washed three times with PBST. 100 μL of SureBlue TMB substrate was added to each well and the plate was incubated for about 10 min at room temperature before stopping the reaction with 100 μL/well of 2N H₂SO₄. The plate was read at 450 nm in a spectrophotometer.

ELISA (GITR Binind, Species Cross-Reactivity, Alanine Scanning)

Binding of the MABs to GITR from various species, various alanine mutant constructs or GITR extracellular domain—was assessed using a 384-well plate was coated with rat, mouse, human or cyno GITR extracellular domain (ECD) at 50 ng per well and incubated overnight at 4° C. The plate was blocked with a solution of 1% BSA in PBS for one hour at room temperature and then rinsed three times with PBST. The MAB was then diluted to 0.5 μg/mL or 1 μg/mL in PBS and 20 μL was added to each well. The plate was incubated for 1 hour at room temperature and then washed three times with PBST. Anti-human kappa antibody (Sigma #A7164), anti-human gamma antibody (Jackson Immunoresearch 109-036-098), goat anti-mouse antibody (Jackson ImmunoResearch 115-035-071) conjugated to HRP was diluted 1:5000 in Blocking Buffer (25 μL) and added to each well, or a hrp conjugated HIS antibody (QIAGEN 1014992) diluted 1:1000 in Blocking Buffer was added. The plate was incubated for 1 hour at room temperature, and then washed six times with PBST. 25 μL of SureBlue TMB (KPL 52-00-02) substrate was added to each well and the plate was incubated for about 10 min at room temperature. Plates were read at 650 nm in a spectrophotometer.

Cell Lines, Cells Cell Lines

To generate the 293-hGITR-NFκB reporter gene cells line, 293 cells were stably transfected with an NFκB-Luciferase reporter gene and human GITR (or cyno GITR). Activation of the GITR signaling pathway in these cells was determined by measuring the levels of luciferase induced within the cells after a 24 hour incubation with GITR-L or agonistic antibody. To assess the effects of cross-linking Abs, they were incubated with an excess of a F(ab′)₂ goat anti-human Fcγ fragment specific antibody or protein A before using in the reporter gene assay.

Clonal Daudi cell lines were generated that express levels of GITR seen on human immune cells.

Cynomolgus monkey PBMCs were prepared and GITR binding determined using MABs. Briefly, cynomolgus blood was transferred into 50 mL conical tubes (Falcon, #352098), then diluted 1:2 in PBS (HyClone, #SH30256.01) and mixed. Diluted blood was carefully layered on top of 18 mL of 90% Ficoll Paque PLUS (GE Healthcare #17-1440-03 diluted in PBS), and tubes were spun at 2,000×g in a bench top centrifuge for 30 minutes at room temperature, with no brake. The plasma layer was carefully removed without disturbing the diffuse PBMC layer on top of the Ficoll. PBMCs were then carefully harvested and PBS was added to the isolated PBMCs until the volume in the conical tube was 45 mL, mixed, and then spun at 300×g in a bench top centrifuge for 15 minutes at room temperature. Supernatant was carefully aspirated and 4 mL of 1×BD Lysing solution (BD #555899) was added and the samples were gently vortexed. After incubating at room temperature in the dark for 3 minutes, 40 mL of PBS was added to each sample and they were spun at 200×g in a bench top centrifuge for 10 minutes at room temperature. Supernatant was carefully aspirated and the pellet was washed two times in 45 mL of PBS before being spun at 200×g in a bench top centrifuge for 10 minutes at room temperature. Resulting pellet was filtered and resuspended at 1×10⁶ cells/mL in CTL Test media (CTLT-005) supplemented with penicillin/streptomycin/glutamine (Hyclone #SV30082.01). 100 μL of purified cynomolgus PBMCs were placed in a 96 well round bottom plate (Corning, #3799). To activate the PBMCs, 100 μL of M-280 Tosylactivated dynabeads (Life Technologies #142.04) conjugated with SP34-2/CD28.2 antibodies was added to each well. A ratio of 3:1 CD3/CD28 Beads to PBMCs was used and the plates were incubated in a 37° C. tissue culture incubator for 48 hours. For day 0 staining 200 μL of PBMCs was placed in a 96-well round bottom plate (Corning, #3799). For samples that were stimulated for 48 hours 100 μL of supernatant was carefully removed and the remaining content of the well was carefully resuspended and 200 μL transferred to the FACS staining plate.

FACS

Plates were prepared with cells resuspended in 200 μL of cold PBS. LIVE/DEAD fixable stain (Life Technologies #L23105) was reconstituted in 50 μL of DMSO and 1 μL of reconstituted stain was added/mL of cold PBS, and cell pellets were immediately resuspended in 100 μL of the LIVE/DEAD PBS solution, incubated for 30 minutes on ice protected from light, then washed and resuspended in 100 μL of cold FACS Buffer containing 2 μg/mL of MAB7 or an Isotype Human IgG1 control antibody and plates incubated for 30 minutes on ice protected from light. Wash and resuspension in 100 μL of antibody cocktail (PerCP Cγ5.5 anti-human CD3 (BD #552852), Alexa Fluor 700 anti-human CD4 (BD #560836), V450 anti-human CD8 (BD #561426), PE-Cγ7 anti-human CD25 (BD #561405) and PE anti-human in FACS Buffer (Jackson Immuno #109-116-098)) followed and plates were then incubated for 30 minutes on ice protected from light and then spun in a bench top centrifuge at 3,200 RPM for 1 minute at 4° C. Cells were washed in FACS Buffer then resuspended in 100 μL of BD CytoFix (BD #554655) and plates were incubated for 15 minutes at room temperature protected from light then washed twice and resuspended in 100 μL of FACS Buffer. Plates were covered with foil (Beckman Coulter, #538619) and stored at 4° C. until ready to read. On the day of FACS read the plate was spun in a bench top centrifuge at 3,200 RPM for 1 minute and 50 μL of CML latex beads (Life Technologies #C37259), 4×10⁵/mL in FACS Buffer, was added to each well. The plates were read on a BD Fortessa flow cytometer and data analyzed using FlowJo.

Transgenic Mice

hGITR knock-in mice were generated by replacing the entire coding sequence (exons and introns) of mouse GITR with the human GITR cDNA sequence. Untranslated sequences upstream of the start codon and downstream of the stop codon are from mouse genome. Gene targeting was done by standard techniques in BALB/c ES cells with targeting vectors bearing BALB/c derived homology arms. Several ES cell clones were identified by PCR and confirmed by Southern blotting to contain the exact human cDNA knockin. Following standard mouse embryology techniques, positive ES cell clones were injected into blastocysts, which were transferred into pseudopregnant recipient foster mothers to derive chimeric offspring. Male chimeric mice were crossed with BALB/c females expressing Cre recombinase in their germline to excise the loxP flanked neomycin resistance cassette. One clone resulted in white offspring indicating germline transmission of the targeted ES cells. Excision of the loxP-flanked cassette was confirmed by PCR genotyping. A subsequent breeding step with BALB/c wt mice removed the Cre recombinase.

hGITRL knock-in mice were generated by replacing mouse the coding portion of exon 1 with the human GITRL cDNA sequence followed by a bovine growth hormone poly-A signal. All ES cell work and mouse embryology were done similar to the procedures described above. hGITR-hGITRL double knock-in mice were generated by intercrossing the two founder lines for 2 generations to produce homozygous double knock-in mice.

Functional Assays

Functional activity of MABs were tested in an NFkB reporter gene assay for agonist activity. MAB was diluted to 6 μg/mL in PBS and incubated for 30 minutes at room temperature in the presence/absence of a 3 fold excess of F(ab′)₂ fragment goat anti-human Fcγ specific crosslinker. Alternatively, MAB was diluted to 6 μg/mL in PBS and incubated for 30 minutes at room temperature in the presence/absence of a 2 fold excess of Protein A. 10 μL of incubated MAB was then added to a 384 well white clear bottomed assay plate. A HEK-293 cell line stably transfected with hGITR and a NFκB reporter gene was diluted to 5×10⁵ cells/mL and 20 μL of the cell suspension was added to each well. The plate was incubated for 24 hours in a 37° C. tissue culture incubator. 30 μL of Cell Bright Glo was added to each well and the plate was read for luminescence on the Acquest.

The ability of MAB to block ligand binding was assessed using HEK293 NFκB reporter parental cells and hGITR stable cells were used in competition binding assays and FACS analysis. Briefly, harvested cells were plated 1×10⁶ cells per mL, 100 μL per well to a 96 well round bottom FACS plate (Corning), then resuspended in 200 μL of cold FACS buffer (1×PBS+1% FBS-HI+0.1% sodium azide) per well. Human GITR ligand titration was prepared from 270 nM to 1.52 pM in FACS buffer at 100 μL per well. Plates were incubated for 1 hour on ice protected from light, cells washed, then prepared 4 nM isotype control or MAB solutions were prepared and added to appropriate wells at 100 μL per well and plates were incubated for 1 hour on ice protected from light, cells washed, then PE-conjugated goat-anti-human detection antibody (Jackson ImmunoResearch) prepared at 1:100 dilution in FACS buffer was added at 100 μL per well and plates were incubated for 30 minutes on ice protected from light. Cells were washed in FACS buffer then cells were fixed with 100 μL per well of BD CytoFix (BD Biosciences) and incubated for further 15 minutes on ice protected from light. Fixed cells were washed twice, resuspeded at a final volume of 150 μL per well of FACS buffer, and samples analyzed within 1 week on a BD Fortessa flow cytometer (BD Bioscience).

Agonist activity of MABs could also be seen on primary T cells that express endogenous levels of GITR via proliferation and cytokine secretion from primary T cells. MABs were conjugated on to M-280 Tosylactivated beads (Invitrogen #142.04) following the manufacturer's instructions. In some experiments agonist CD3 (OKT3) and CD28 (CD28.2) antibodies were also conjugated to beads. 1×10⁵ freshly purified CFSE-labelled human PBMCs were plated in 10 uL of CTL Test media (CTL #CTLW-010) into a 96 well round bottomed tissue culture plate. 100 uL of MAB conjugated beads was then added at a ratio of 1 T cell: 1 beads. Plates were then incubated for 3 days in a 37° C. tissue culture incubator. Levels of secreted cytokines in the media were measured using a MSD multiplex assay according to manufacturer's instructions. Cells were stained with anti-CD4, -CD8a, -CD25, -GITR antibodies and a LIVE/DEAD stain after staining cells were fixed and read on the flow cytometer. Proliferation of each CD4 and CD8 cells was assessed by CFSE staining and counting beads were added prior to the FACS read to allow normalization of the samples.

Co-stimulatory activity of MABs on T cells was also measured using an ELISpot method for detection of IFNg. Briefly, ELISPOT plates (Millipore MSIPS4510) were prepared by coating with 70% ethanol for 2 minutes followed by PBS wash and incubation overnight with 50 ug IFNg monoclonal antibody in PBS (Mabtech 3321-3). Purified CD8+ T cells were isolated from spleens of Vehicle or MAB7-treated mice 15-days post-dose. T cells were plated into the coated ELISPOT plates at 0.25×10̂6 cells per well in CTL media (CTL Test-medium (CTL CTLT-005), 1 mM Hepes (Mediatech MT25-060-C1), 2 mM L-glutamine (Mediatech MT25-005-C1), 1 mM sodium pyruvate (Mediatech MT25-000-C1), 100 uM MEM Non-essential amino acids (Mediatech MT25-025-C1), 66 uM 2-Mercaptoethanol (Gibco 21985-023), 100 U/mL Pten/Strep (Gibco 10378016). Colon26 cells were treated with 10% ConA sup (BD Biosciences 354115) at 37° C. for 48 hours to upregulate MHC Class II expression and washed with CTL media prior to addition to T-cells. Colon26 tumor cells (20,000/well) were added to CTLs and incubated at 37° C. for 24-48 hours. Plates were then washed with 0.05% Tween-20/PBS and 10 ug of biotinylated anti-IFNg Mab (Mabtech R4-6A2-biotin) was added to each well and incubated for 2 hours at 37° C. Plates were then washed with 0.05% Tween-20/PBS and Vectastain ABC solution (Vector Labs PK6100) was added to each well and incubated for 1 hour at room temperature. Cells were then washed with 0.05% Tween-20/PBS and AEC substrate, prepared according to manufacturer's protocol (Sigma A6926) was added to each well and incubated for 4 minutes at room temperature. Plates were then rinsed with tap water, dried and stored in the dark for 24 hours prior to reading.

The ability of MABs to induce ADCC was measured using a reporter assay. In a 96 well white plate (Perkin Elmer F6178) 2×10³ hGITR-Daudi cells were incubated with 4×10⁴ Jurkat-V158 cells (stably expresses the V158 variant of the human FcgRIIIa and an NFAT reporter) at a ratio of 1 Daudi cell to 20 Jurkat) cells in 50 uL of RPMI+10% FBS. An equal volume of MAB was added to the well and the plates was incubated for 3 hours in a 37° C. tissue culture incubator. After the incubation 60 uL of Bright-Glo was added to each well and the plate was read on a luminometer.

In vitro splenocyte assays were carried out using spleens isolated from mice. Briefly, spleens from mice were dissociated by automated homogenization in 5 mL AutoMACS Rinsing Solution (Miltenyi Biotech 130-091-222) containing 5% BSA (Miltenyi Biotech 130-091-376) using gentleMACS C tubes (Miltenyi Biotech 130-096-334) on a gentleMACS Octo Dissociator (Miltenyi Biotech 130-095-937). Homogenates were strained through a 0.70 uM pore size cell strainer (Fisher Scientific 22363548) and washed with 10 mL AutoMACS buffer. Splenocytes were resuspended and plated at 100,000 cells/well in RPMI (HyClone SH30027.02)+10% human serum (Cellgro 35-060.C1)+1× Pen/Strep/L-Glut (Gibco 15 140-112) in a 96-well tissue culture plate (Costar 3799). For T-cell stimulation, 0.4 ug/mL of anti-mouse CD3 (eBioscience 16-0031-86) and 0.8 ug/mL of anti-mouse CD28 (eBioscience 13-0281-86) antibodies were added to appropriate wells. After 48 hours, cells were either analyzed immediately or pulsed with control or therapeutic antibody for 30 minutes to 96 hours, stained with fluorophore-conjugated antibodies and analyzed by flow cytometry.

Flow cytometry: For surface markers, cells were stained with anti-CD19 (BD Biosciences 562291), anti-CD8 (Biolegend 100725), anti-CD4 (eBioscience 25-0041), anti-CD69 (BD Biosciences 561238), anti-hGITR (Miltenyi Biotech 130-092-895) and anti-hIgG (Life Sciences A-10631) antibodies for 30 minutes at 4C. For intracellular staining, following incubation with cell surface antibodies, cells were washed, fixed and permeabliized with FOXP3 Fix/Perm buffer (Biolegend 421403) according to manufacture's protocol and incubated with anti-phospho-IKKa/b antibody (Cell Signaling 2697) or anti-FOXP3 antibody (eBioscience 50-4774-42) for 30 minutes at 4C. Cells were read on a BD LSRFortessa cytometer using BD FACSDiva software (BD Biosciences) and flow data was analyzed using FlowJo software (TreeStar Inc.).

Single cell suspensions were generated from tumors and spleens and stained for analysis by flow cytometry. For example, cell markers were assessed using the following antibodies: a-CD8-BUV395 (clone 53-6.7, BD Biosciences 563786), a-CD19-APC-Cy7 (clone 6D5, BioLegend 115530), a-CD3-PerCp (clone 145-2C11, BD Bioscience 553067), a-PD-1-PE-Cy7 (clone RMP1-30, Biolegend 109110). Flow cytometry was run on BD LSRFortessa cytometers using BD FACSDiva software (BD Biosciences). Cytometry data was analyzed using FlowJo Software (FlowJo LLC). Graphs were generated and statistics run using Prism software (GraphPad Software). All data were shown as mean±standard deviation (SD). Group comparisons were carried out using student's T-test with two-tailed 95% confidence interval. For all statistical evaluations the level of significance was set at p<0.05. Significance compared to the vehicle control group is reported unless otherwise stated.

In vivo tumor models. The Colon26 murine colon carcinoma cell line was obtained from the Division of Cancer Treatment and Diagnosis at the National Cancer Institute (vial: 0507238). Murine Colon26 carcinoma cells were cultured in RPMI 1640 medium (HyClone SH30027.02) supplemented with 10% FBS (Gibco 10099-141), 10 mM HEPES (Gibco 15630-080) and 1 mM sodium pyruvate (Gibco 11360-070). 8-10 week old female hGITR-hGITRL knock-in mice were injected subcutaneously with 0.5×10̂6 Colon26 cells in 100 uL of RPMI in the flank. Tumors were measured using digital calipers and tumor volume calculated using the equation (L×W²)/2. When tumors reached an average size of 180 mm³, mice were randomized and dosed with a single intraperitoneal injection of vehicle (PBS) or therapeutic antibody (15 mg/kg) in 200 uL PBS. Mice were sacrificed and tumors collected for analysis by flow cytometry 7 days after dosing with therapeutic antibodies. All animal experiments were performed in an AAALAC accredited facility using IACUC approved protocols. Statistical analysis was performed in Prism software using student's t-test with two-tailed 95% confidence interval or One-way ANOVA with Tukey correction.

Surrogate murine GITR Colon26 model testing. Charles River Labs female 6-8 week old BALB/c mice were used as the experimental animal. For implantation, cells were resuspended in Hank's 1× Balanced Salt Solution (Hyclone Cat# SH30030.02) and implanted with a subcutaneous injection into the right lower flank using a 28 g needle (100 ml injection volume). After implantation, mice were randomized according to tumor volume. Mice were dosed with 5 mg/kg of IgG2a-DTA-1 or mouse IgG2a isotype control by subcutaneous injection. Clone DTA-1, a rat anti-mouse GITR antibody (S. Sakaguchi, Kyoto University, Kyoto Japan) was modified to create a murine chimeric IgG2a by fusing the DTA-1 variable region sequences to murine IgG2a Fc regions to create IgG2a-DTA-1.

Combination Therapy. To assess in vivo activity of surrogate anti-GITR antibody (mouse IgG2a-DTA-1), in combination with surrogate anti-PD-1 antibody (rat, IgG2a RMP1-14, Biolegend), female 6-8 weeks old BALB/cJ mice from Jackson Laboratories (Bar Harbor, Me.) were implanted subcutaneously in the right supra-axillary region with 5×10⁵ Colon26 cells in a volume of 100 uL. For implantation, Colon26 cells were suspended in Dulbecco's PBS, calcium and magnesium free from Lonza (17-512F). Mice were enrolled in the study ten days post implantation with a mean±SEM tumor volume of 115 mm³±7. After being randomly assigned to one of 4 groups (n=10-16/group), mice were dosed concurrently once weekly for 2 weeks with isotype (group1), RMP1-14 (10 mg/kg, group2), IgG2a-DTA-1 (5 mg/kg, group3) or the combination of RMP1-14 and IgG2a-DTA (10 mg/kg and 5 mg/kg, respectively, group 4) as described in Table 6. Day 0 is defined as the day of randomization. The isotype control group contained mIgG2a (Biolegend) at 5 mg/kg and rat IgG2a (Biolegend) at 10 mg/kg. IgG2a-DTA and its isotype control (mIgG2a) were dosed via subcutaneous injection at 5 mg/kg. RMP1-14 and its isotype control (rIgG2a) were dosed via intraperitoneal injection. Dosing volume was 10 mg/mL for all treatments. Body weights and tumor volumes were collected two-three times per week. Individual animals were scored as achieving end point when tumor volumes equaled or exceed 1200 mm³. Anti-tumor activity was reported based on changes in the median time to endpoint (TTE), assessed by Kaplan-Meier survival analysis.

Combination Therapy. To assess expression of costimulatory molecules following administration of anti-GITR or anti-GITR in combination with anti-PD-1, single cell suspensions of whole tumors and spleens were profiled by flow cytometry following 1 dose of a GITR (clone IgG2a-DTA-1) or anti-PD1 (clone RMP1-14) or anti-GITR+PD1 in combination. mIgG2a was used as control. LAG3, TIM3 and PD-1 positive cells were assessed as a percentage of total CD3+CD8+ T cells in tumors and spleens. Pvalues are calculated with t-test. *p<0.05 and **p<0.005.

Results Murine and Reference V-Region Amino Acid Sequences

RT-PCR products from hyrbidoma cells that express MAB1 were sequenced, and this sequence was largely (95% or greater) verified at the protein level using a ThermoElectron LTQ-Orbitrap Mass Spectrometer. The heavy and light chain variable regions of MAB1 were then cloned in KaloBios vectors in order to create the reference Fab MAB1rFab. The first amino acid in MAB1 has to be changed from an asparagine (N) to a glutamic acid (E) residue to enable cloning into KaloBios vectors for generation of the reference Fab; therefore, the MAB1rFab has glutamic acid at the first VK position. The Fab MAB1rFab has intact murine V-regions from MAB1 fused with human constant regions. In addition to MAB1rFab, an optimized Fab, MAB1opFab, was constructed. Several framework amino acid residues in MAB1rFab were changed to human germline residues in MAB1opFab.

Reference and Optimized Reference Fab Affinity Analysis

Human germline residues incorporated into the optimized reference MAB1opFab in FR1 and FR3 are those specified by the PCR primers used to amplify the human V-segment repertoire and thus are present in all members of the Humaneered® Antibody V-region libraries. The optimized reference Fab is constructed to assess whether or not any of the changes to human germline alter the properties of Fab binding. Affinity constants (Ka (1/Ms), Kd (1/s), and KD (M) of MAB1rFab, MAB1opFab was assayed using the ForteBio Octed QK system and Striptavidin High Binding Biosensors coated with biotinylated hGITR-hFc. Compared with MAB1rFab, MAB1opFab, had very similar Kd, but five-fold improvement on Ka indicating that the amino acid changes in MAB1rFab are tolerated.

Library Construction and Selection of Humaneered® Antibody Fabs

Heavy and light chain front-end and FR3 cassette libraries, germline-family restricted to VH3 and VKIII, were generated and screened by CLBA. For VK, clones that support binding to human GITR were identified from both VK front-end (MAB1VK3FE-01) and FR3 (MAB1VK3FR3-01) cassette libraries. For VH, clones that support binding to human GITR were identified from FR3 cassette library (MAB1VH3FR3-01), but not from the VH3 front-end library (MAB1VH3FE-01). Since the majority members in Vk front-end and FR3 cassette libraries were positive in CLBA, the whole repertoire of these two libraries was used to construct Vk full-chain library (MAB1VK3FcL-01) by overlapping PCR with mutagenic VK CDR2s that encodes either the parental murine or the selected human germline (VKIII L-16) residue at all positions. A number of hGITR positive clones were identified from VH3FR3 library (MAB1VH3FR3-01) through CLBA and confirmed by human GITR specific ELISA. Six of them were used to pair with VK full-chain library (MAB1Vk3FcL-01) to enable functional Fab expression and the screen of this library.

Since there were no clones that bind hGITR with high affinity were identified from VH front-end library (MAB1VH3FE-01), subsequently, a second VH3 front-end library (MAB1VH3FE-02) was constructed. This library has either the parental murine or human germline (VH3 3-30) residue at each position of CDR1 and the FR3 sequences from the six selected VHFR3 clones. Many hGITR binders were identified from both VK full-chain library (MAB1Vk3FcL-01) and the second VH front-end library (MAB1VH3FE-02). These clones were confirmed by human GITR specific ELISA assay on Fab-containing cell supernatants and rank-ordered by hGITR affinity titration ELISA.

Based on hGITR affinity titration ELISA, four VK full-chain clones were selected from VK full-chain library (MAB1VK3FcL01), and six clones were selected from MAB1VH3FE-02 library. The six VH clones were used as the backbone to construct the VH full-chain library with either MAB1 murine or the closest human germline (VH3 3-30) residue at each position in CDR2. This VH full-chain repertoire was paired with the four VK full-chain clones to form the final human full-chain Fab library. CLBA identified many hGITR binding clones, that were confirmed by ELISA using the respective culture supernatant as the Fab source. Five human full-chain Fab clones (MAB2, MAB3, MAB4, MAB5, and MAB6) were selected based on DNA sequence analysis and hGITR affinity titration ELIS A results.

Testing the Affinity of Humaneered® Antibody Fabs for GITR Antigen Using ForteBio Octet Analysis

The five human full-chain Fabs (MAB2, MAB3, MAB4, MAB5, and MAB6) were expressed and purified. The binding kinetics of these human Fabs were then compared to the kinetics of the optimized reference Fab (MAB1opFab) using the ForteBio Octet system (numerical data summarized in Table 3).

TABLE 3 Affinity of Fabs for human GITR Fab KD [M] MAB1opFab (a)* 1.25E−8 MAB2 (a) 6.84E−9 MAB3 (a) 2.98E−9 MAB1opFab (b)* 6.59E−9 MAB4 (b) 2.43E−9 MAB5 (b) 3.74E−9 MAB1opFab (c)* 1.47E−8 MAB6 (c) 5.94E−9 *a, b, c indicate three separate Forte experiments. The results are global fitting of two sample duplicates.

Amino Acid Sequence of Antibodies MAB2, MAB3, MAB4, MAB5, MAB6, and Percentage Identity to Human Germline Sequence

The variable region amino acid sequences of the five Fabs (MAB2, MAB3, MAB4, MAB5, MAB6,) are shown in Table 1. The percent identity to human germline sequences of the five Fabs was determined by aligning the Vh and Vk amino acid sequences against a single germline sequence (VKIII L16/A27 and VH3 3-30, respectively; Table 4). Residues in CDRH3 and CDRL3 were omitted from the calculation for each chain.

TABLE 4 Percent identity of the five Fabs to human germline sequences Fab VKIII L15/A27 VH3 3-30 Fv MAB2 95% 86% 90% MAB3 98% 85% 91% MAB4 95% 85% 89% MAB5 95% 83% 89% MAB6 95% 82% 88% MAB7 95% 85% 89% MAB8 95% 85% 89%

Conservation of Human GITR Antigenic Epitope

Antigen epitope conservation was evaluated by an indirect Competition ELISA. All five Fabs blocked the parental mouse antibody MAB1 binding to human GITR indicating that these human Fabs retain the epitope specificity of the original murine antibody.

Analysis of Antigen Specificity of MAB4 and MAB5 by ELISA

In order to test whether antigen specificity of the parental mouse antibody MAB1 was retained in the IgGs, MAB2, MAB3, MAB4 and MAB5, binding of the antibodies to a panel of human TNFRs was tested in an ELISA assay, The results of one such assay with MAB4 and MAB5 (FIG. 2C) show that MAB4 and MAB5 retain high specificity for GITR, similar to the murine antibody MAB1. Similar results were obtained with MAB2, MAB3 and MAB6.

Antibody Binding to Human and Cynomolgus Macaque but not Rodent GITR Protein in ELISA

The parental mouse antibody MAB1 binds to human and cynomolgus but not rodent GITR protein. FIG. 2A-B shows that, like MAB1, antibodies MAB4 and MAB5 were able to bind in a similar manner both human and cynomolgus GITR, but not rodent GITR. Similar results were found with MAB6, 7, and 8.

Binding affinities of GITR agonist antibodies MAB4 and MAB5 for human (hGITR) and cyno (cGITR) GITR, were determined by Biacore analysis. See Table 5. Monoclonal antibodies MAB4 and MAB5 bind to human GITR with subnanomolar binding affinities (KD). Antibodies MAB4 and MAB5 bind to cyno GITR with nanomolar binding affinities that are about 2-3 fold lower than the binding affinities for human GITR. The anti-GITR agonist antibodies of the invention bind selectively to human and cyno GITR in a number of biochemical assays, including flow cytometry, ELISA, Biacore, and Protagen™ chip assays.

TABLE 5 Binding affinities of MAbs to human- and cyno- GITR Antigen mAb KD (nM) hGITR MAB4 0.684 (±0.331) hGITR MAB5 0.973 (±0.167) hGITR MAB7 4.29 (±0.14) cGITR MAB4  1.78 (±0.543) cGITR MAB5  1.87 (±0.520) cGITR MAB7 3.67 (±0.09)

Monoclonal antibody MAB7 binds to human as well as cyno CD4+ Tcells. FACS analysis of isolated cyno or human PBMCs demonstrated MAB7 binds isolated CD4+ Tcells. Additionally, FACS experiments demonstrated GITR (by binding of MAB7) and CD25 upregulation following CD3/CD28 activiation of PBMCs (CD4+ Tcells). (data not shown)

Functional Activity of Antibodies and in Reporter Gene Assays and Cell Assays

Antibodies were assayed in a reporter gene assay for functional activity (FIG. 3). Each of MAB4, MAB5, MAB7 and MAB8 IgGs induce NFκB activity when crosslinked, at levels similar to GITR ligand (GITR-L). See FIG. 3A-D. Similar results were obtained with MAB2, MAB3, and MAB6 (data not shown).

MAB7 competes with human GITR ligand for binding human GITR expressing stable cell line. Competition assays were performed in triplicate sets of values, FACS competition analysis demonstrates inhibition of ligand binding. See FIG. 2D.

To confirm functional activity on endogenous GITR, antibodies were conjugated to beads and incubated with purified CFSE labelled human PBMCs. MAB7 induces an increase in proliferation of both CD4+ T cells (FIG. 4A) and CD8+ T cells (FIG. 4B) compared to an isotype control antibody. This increase in proliferation was also accompanied by an increase in the secretion of several cytokines, including IFNγ (FIG. 4C), TNFα, IL-10 and IL-13 (not shown). Similar results were found with MAB 4, MAB5 (not shown). We were able to show that the increase in proliferation and IFNγ production induced by MAB7 was dependent upon the presence of anti-CD3 and anti-CD28 agonistic antibodies on the beads. If these co-stimulatory antibodies were omitted MAB had no agonist effects on either CD4+ or CD8+ T cells. Similar results were obtained with MAB2, MAB3, MAB4; MAB5 and MAB6.

MAB7 was also found to demonstrate capability to induce FcgRIIIa signaling (shown to be correlated with ADCC activity) in an in vitro assay when high levels of GITR are present. Daudi-hGITR cells incubated with MAB7 or control Ab, and the Jurkat-V158 cell line showed MAB7 is able to induce FcgRIIIa signaling in an in vitro assay and that the ability of MAB7 to induce FcgRIIIa signaling correlates with the receptor level expressed on the surface of the Daudi cells (i.e. higher receptor levels equals greater FcgRIIIa signaling induction). See FIG. 5.

hGITR is expressed on T-cells and is functional in hGITR-hGITRL knock-in mice. Splenocytes were isolated from wild type or hGITR-hGITRL knock-in mice and cultured either without stimulation or with stimulation using a-CD3 and a-CD28 antibodies for 24, 48, 72 or 96 hours. Cells were then stained with fluorophore-conjugated antibodies and analyzed by flow cytometry, demonstrating human GITR is expressed, and costimulation results in increased GITR expression profile in wild type or transgenic mice. Splenocytes isolated from hGITR-hGITRL knock-in mice demonstrate induction of GITR expression in response to costimulation in culture (FIG. 6A). MAB7 effectively binds hGITR expressed on CD8+ cells (FIG. 6B); and MAB7 binding to stimulated Tcells correlates with increased Tcell activation as measured by pIKK staining (FIG. 6C) and T cell activation marker CD25+(FIG. 6D).

MAB7 is functional in vivo. hGITR-hGITRL double knock-in mice with established Colon26 tumors were treated with a single dose of vehicle (n=8/timepoint) or MAB7 (n=10/timepoint) antibody as described above. Tumors were measured twice per week and tumor volume calculated using the equation (L×W²)/2. MAB treated animals demonstrated delayed growth of Colon26 tumors. At three days post treatment, whole blood (FIG. 7B-7C) and tumors (FIG. 7D-7E) were collected and analyzed by flow cytometry for cell surface hGITR expression on immune cells. Results suggest hGITR occupancy and shedding resulting in decreased hGITR from treated groups for both Tregulatory cells and Thelper cells in both blood and tumors (*p<0.05, ****p<0.00005).

MAB7 elicits an anti-tumor immune response to Colon26 tumors in vivo. hGITR-hGITRL double knock-in mice with established Colon26 tumors were treated with a single dose of vehicle (n=8/timepoint) or MAB7 (n=10/timepoint). FIG. 8A depicts results 3-days post-dose, demonstrating Tregs are reduced in treated animals. FIG. 8B-8C depict results 15-days post-dose, demonstrating increased lymphocytes (8B) and increased activated CD8+ T cells (8C) present in tumor site following treatment. The absolute number of cells was normalized to tumor size to account for the significant difference in tumor size between Vehicle and MAB7 treated groups. MAB7 results suggest treatment results in increased Teff/Treg ratio in treated animals as determined by total intratumoral activated CD8+ T cells compared to CD4+ FOXP3+ Tregs. See FIG. 8D. Additionally, results of splenocyte assays from purified CD8+ T cells incubated with Colon26 tumor cells ex-vivo, and measuring CTL response using IFNg ELISPOT assay suggest increased tumor specific IFNg response in CD8+ T cells from MAB7 treated animals. (*p<0.05, ***p<0.0005). See FIG. 8E.

Treatment of mice with anti-mGITR Ab upregulates PD-1 expression in tumors and spleen. Mice with established Colon26 tumors were treated with two doses of control or murine anti mGITR antibody. FIG. 9A9C depicts results demonstrating PD-1 expression is upregulated on CD8+ T cells in Colon26 tumors as well as spleens after treatment with surrogate GITR antibody, IgG2a-DTA-1.

GITR and PD-1 combinations confer survival advantage compared to isotype control. Anti-GITR (DTA-1) and anti-PD-1 (RMP1-14) were administered alone and in combination in mice with established Colon26 tumors. See FIG. 10. Combination administration shows significant survival advantage compared to isotype control (***p<0.0005 pairwise comparison using the Gehan-Breslow-Wilcoxon test). Anti-mGITR (IgG2a-DTA-1) single agent shows significant survival advantage compared to isotype control (*p<0.05 pairwise comparison using the Gehan-Breslow-Wilcoxon test). The data indicate that the combination of IgG2a-DTA-1 and RMP1-14 confers a statistically significant survival advantage relative to isotype control treatment with a median TTE greater than 42 days (median TTE not achieved) (P<0.0005) relative to 22 days for the isotype treated group. Notably, 3/10 animals achieved a complete regression (CR), 2/10 animals achieved stable disease (SD). IgG2a-DTA-1 monotherapy resulted in a median TTE of 30.5 days (P<0.05), with 3/10 animals achieving stable disease (SD). The median survival of the RMP1-14 treated group was 24 days, which was not statistically significantly different from the isotype treated group. Kaplan Meier Graphs were generated and statistics performed using Prism software (GraphPad Software). Group comparisons were carried out as pairwise comparison using the Gehan-Breslow-Wilcoxon test. For all statistical evaluations the level of significance was set at p<0.05. Significance compared to the vehicle control group is reported. Stable disease is defined as 3 successive tumor volume measurements with 10% or less change in tumor volume.

TABLE 6 Combination Therapy Group Ab1 (5 mg/kg, SC) Ab2 n/group 1 mIgG2a rIgG2a (10 mg/kg, IP) n = 10 2 RMP1-14 (10 mg/kg, IP) mIgG2a (5 mg/kg, SC) n = 16 mIgG2a 3 DTA-1 rIgG2a (10 mg/kg, IP) n = 10 4 DTA-1 RMP1-14 (10 mg/kg, IP) n = 10 IP = intraperitoneal; SC = subcutaneous

Expression of costimulatory molecules was assessed in tumors following administration of anti-GITR or anti-GITR in combination with anti-PD-1. See FIG. 11. Results of single cell suspensions of whole tumors and spleens profiled by flow cytometry following 1 dose of anti-GITR or anti-PD1 or anti-GITR+PD1 in combination demonstrated increased expression of LAG3, TIM3 and PD-1 on CD8+ T cells in Colon26 tumors after treatment with GITR, PD-1 and in combination. A single combination dose demonstrated upregulated expression of PD-1 in spleen CD8+ cells.

INCORPORATION BY REFERENCE

All publications, patents, and Accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. 

1.-75. (canceled)
 76. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an anti-GITR agonist antibody or antibody fragment thereof in combination with one or more additional therapeutic agents, wherein the anti-GITR agonist antibody or antibody fragment comprises (a) a heavy chain variable region (VH) comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of a sequence selected from any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109; and a light chain variable region (VL) comprising: a VLCDR1 of SEQ ID NO:30 or SEQ ID NO:31, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; or (b) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109, and a VL comprising: a VLCDR1 of SEQ ID NO:85 or SEQ ID NO:86, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83, and wherein the one or more additional therapeutic agents are selected from any of the following: a STING agonist, a TLR agonist, an A2AR antagonist, or an oncolytic virus, a vascular endothelial growth factor receptor (VEGFR) inhibitor, a c-Met inhibitor, a TGFβ inhibitor, an IDO/TDO inhibitor, a vaccine, a bi- or tri-specific cell engager, an inhibitor of IAP (Inhibitor of Apoptosis Protein), an inhibitor of EGFR (Epidermal Growth Factor Receptor), an inhibitor of target of rapamycin (e.g., mTOR), IL-15 or a variant thereof, a CTLA-4 inhibitor, a bispecific antibody molecule that binds to CD3 and a tumor antigen, a PD-1 inhibitor, another GITR agonist, an inhibitor of an immune checkpoint molecule selected from PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β, a CSF-1/1R inhibitor, an IL-17 inhibitor, an IL-1β inhibitor, a CXCR2 inhibitor, an inhibitor of PI3Kγ or PI3Kδ, a BAFF-R inhibitor, a MALT-I/BTK inhibitor, a JAK inhibitor, a CRTH2 inhibitor, a PFKFB3 inhibitor, or an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83 ligand, a chemotherapeutic agent, or a cytotoxin.
 77. The method of claim 76, wherein the additional therapeutic agent is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.
 78. The method of claim 76, wherein the additional therapeutic agent is an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.
 79. The method of claim 76, wherein the additional therapeutic agent is an antagonist of CTLA4, LAG3, or TIM3, or an inhibitor of PD-1/PD-L1 interaction.
 80. The method of claim 76, wherein the cancer is selected from epithelial cancers, carcinomas, sarcomas or lymphomas.
 81. The method of claim 76, wherein the cancer is selected from renal cancer, lung cancer including non-small cell lung cancer (NSCLC), glioma, fibrosarcoma, pancreatic cancer, melanomas, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, or head and neck squamous cell carcinoma (HNSCC).
 82. The method of claim 76, wherein the antibody or antibody fragment comprises a heavy chain variable segment that has at least 95% sequence identity to SEQ ID NO:16, and a light chain variable segment that has at least 95% sequence identity to SEQ ID NO:17.
 83. The method of claim 76, wherein the antibody or antibody fragment comprises a heavy chain comprising SEQ ID NO:16 and a light chain comprising SEQ ID NO:17.
 84. The method of claim 76, wherein the heavy chain framework region 4 (FR4) of the antibody or antibody fragment is a human germline FR4 comprising SEQ ID NO:42.
 85. The method of claim 76, wherein the light chain FR4 of the antibody or antibody fragment is a human germline FR4 comprising SEQ ID NO:50.
 86. The method of claim 76, wherein the antibody or antibody fragment comprises: (a) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:23, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:30, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; (b) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:85, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83; (c) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:24, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:31, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; (d) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:86, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83; (e) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:25, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:30, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; (f) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:26, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:30, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; (g) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:85, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83; (h) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:27, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:30, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; (i) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:25, and a VHCDR3 of SEQ ID NO:109, and a VL comprising: a VLCDR1 of SEQ ID NO:30, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; or (j) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, a VHCDR3 of SEQ ID NO:109, and a VL comprising: a VLCDR1 of SEQ ID NO:85, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83.
 87. The method of claim 76, wherein the antibody or antibody fragment comprises a VH comprising an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 99 or
 105. 88. The method of claim 76, wherein the antibody or antibody fragment comprises a VH comprising an amino acid sequence selected from any one of SEQ ID NOs: 6, 8, 10, 12, 14, 99, or
 105. 89. The method of claim 76, wherein the antibody or antibody fragment comprises a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 7 or SEQ ID NO:
 9. 90. The method of claim 76, wherein the antibody or antibody fragment comprises a VL comprising an amino acid sequence selected from SEQ ID NO: 7 or SEQ ID NO:
 9. 91. The method of claim 76, wherein the antibody or antibody fragment comprises: (a) a VH comprising SEQ ID NO:6, and a VL comprising SEQ ID NO:7; (b) a VH comprising SEQ ID NO:8, and a VL comprising SEQ ID NO:9; (c) a VH comprising SEQ ID NO:10, and a VL comprising SEQ ID NO:7; (d) a VH comprising SEQ ID NO:12, and a VL comprising SEQ ID NO:7; (e) a VH comprising SEQ ID NO:14, and a VL comprising SEQ ID NO:7; (f) a VH comprising SEQ ID NO:99, and a VL comprising SEQ ID NO:7; and (g) a VH comprising SEQ ID NO:105, and a VL comprising SEQ ID NO:7.
 92. The method of claim 76, wherein the antibody or antibody fragment comprises a VH comprising SEQ ID NO:99, and a VL comprising SEQ ID NO:7.
 93. The method of claim 76, wherein the antibody comprises a heavy chain comprising an amino acid sequence selected from any one of SEQ ID NOs: 65, 69, 73, 75, 77, 100, or 106; and a light chain comprising an amino acid sequence selected from SEQ ID NO:66 or SEQ ID NO:70.
 94. The method of claim 76, wherein the antibody comprises: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 65, and a light chain comprising the amino acid sequence of SEQ ID NO: 66; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 69, and a light chain comprising the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 73, and a light chain comprising the amino acid sequence of SEQ ID NO: 66; (d) a heavy chain comprising the amino acid sequence of SEQ ID NO:75, and a light chain comprising the amino acid sequence of SEQ ID NO: 66; (e) a heavy chain comprising the amino acid sequence of SEQ ID NO: 77, and a light chain comprising the amino acid sequence of SEQ ID NO: 66; (f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100, and a light chain comprising the amino acid sequence of SEQ ID NO: 66; or (g) a heavy chain comprising the amino acid sequence of SEQ ID NO: 106, and a light chain comprising the amino acid sequence of SEQ ID NO:
 66. 95. The method of claim 76, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100; and a light chain comprising the amino acid sequence of SEQ ID NO:66.
 96. The method of claim 76, wherein the antibody or antibody fragment is humanized.
 97. The method of claim 76, wherein the antibody fragment is a Fab, Fab′, F(ab′)2, Fd, Fv, or a single chain Fv fragment (scFv).
 98. The method of claim 76, wherein the antibody or antibody fragment comprises a heavy chain constant region of human IgG1, and a light chain constant region of human kappa chain.
 99. The method of claim 76, wherein the antibody or antibody fragment is cross-linked to a second anti-GITR antibody or antibody fragment.
 100. The method of claim 76, wherein the antibody or antibody fragment induces an elevated Teff:Treg ratio in vivo.
 101. The method of claim 76, wherein the antibody or antibody fragment induces a potentiated immune response in vivo.
 102. A method of treating an infectious disease in a subject in need thereof, the method comprising administering to the subject an anti-GITR agonist antibody or antibody fragment thereof in combination with one or more additional therapeutic agents, wherein the anti-GITR agonist antibody or antibody fragment comprises (a) a heavy chain variable region (VH) comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of a sequence selected from any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109; and a light chain variable region (VL) comprising: a VLCDR1 of SEQ ID NO:30 or SEQ ID NO:31, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; or (b) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109, and a VL comprising: a VLCDR1 of SEQ ID NO:85 or SEQ ID NO:86, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83, and wherein the one or more additional therapeutic agents are selected from any of the following: a STING agonist, a TLR agonist, an A2AR antagonist, or an oncolytic virus, a vascular endothelial growth factor receptor (VEGFR) inhibitor, a c-Met inhibitor, a TGFβ inhibitor, an IDO/TDO inhibitor, a vaccine, a bi- or tri-specific cell engager, an inhibitor of IAP (Inhibitor of Apoptosis Protein), an inhibitor of EGFR (Epidermal Growth Factor Receptor), an inhibitor of target of rapamycin (e.g., mTOR), IL-15 or a variant thereof, a CTLA-4 inhibitor, a bispecific antibody molecule that binds to CD3 and a tumor antigen, a PD-1 inhibitor, another GITR agonist, an inhibitor of an immune checkpoint molecule selected from PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β, a CSF-1/1R inhibitor, an IL-17 inhibitor, an IL-1β inhibitor, a CXCR2 inhibitor, an inhibitor of PI3Kγ or PI3Kδ, a BAFF-R inhibitor, a MALT-I/BTK inhibitor, a JAK inhibitor, a CRTH2 inhibitor, a PFKFB3 inhibitor, or an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand.
 103. The method of claim 102, wherein the additional therapeutic agent is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.
 104. The method of claim 102, wherein the additional therapeutic agent is an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.
 105. The method of claim 102, wherein the additional therapeutic agent is an antagonist of CTLA4, LAG3, or TIM3, or an inhibitor of PD-1/PD-L1 interaction.
 106. The method of claim 102, wherein the infectious disease is selected from a bacterial infection, a fungal infection, a viral infection or a parasitic infection.
 107. A method of enhancing a T cell response in a subject in need thereof, comprising administering to the subject an anti-GITR agonist antibody or antibody fragment thereof in combination with one or more additional therapeutic agents, wherein the anti-GITR agonist antibody or antibody fragment comprises (a) a heavy chain variable region (VH) comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of a sequence selected from any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109; and a light chain variable region (VL) comprising: a VLCDR1 of SEQ ID NO:30 or SEQ ID NO:31, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; or (b) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109, and a VL comprising: a VLCDR1 of SEQ ID NO:85 or SEQ ID NO:86, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83, and wherein the one or more additional therapeutic agents are selected from any of the following: a STING agonist, a TLR agonist, an A2AR antagonist, or an oncolytic virus, a vascular endothelial growth factor receptor (VEGFR) inhibitor, a c-Met inhibitor, a TGFβ inhibitor, an IDO/TDO inhibitor, a vaccine, a bi- or tri-specific cell engager, an inhibitor of IAP (Inhibitor of Apoptosis Protein), an inhibitor of EGFR (Epidermal Growth Factor Receptor), an inhibitor of target of rapamycin (e.g., mTOR), IL-15 or a variant thereof, a CTLA-4 inhibitor, a bispecific antibody molecule that binds to CD3 and a tumor antigen, a PD-1 inhibitor, another GITR agonist, an inhibitor of an immune checkpoint molecule selected from PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β, a CSF-1/1R inhibitor, an IL-17 inhibitor, an IL-1β inhibitor, a CXCR2 inhibitor, an inhibitor of PI3Kγ or PI3Kδ, a BAFF-R inhibitor, a MALT-I/BTK inhibitor, a JAK inhibitor, a CRTH2 inhibitor, a PFKFB3 inhibitor, or an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand.
 108. The method of claim 107, wherein the additional therapeutic agent is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.
 109. The method of claim 107, wherein the additional therapeutic agent is an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.
 110. The method of claim 107, wherein the additional therapeutic agent is an antagonist of CTLA4, LAG3, or TIM3, or an inhibitor of PD-1/PD-L1 interaction. 